Telomerase Inhibitors

ABSTRACT

Methods and compositions for treating cancer and other diseases in which inhibition of telomerase activity can ameliorate disease symptoms or prevent or treat the disease relate to compounds that are derivatives of benzo b!thiophenes. Such compounds are characterized by the following structure: ##STR1## In this compound, R 1  is selected from the group consisting of --OR 7 , --NR 8  R 9 , --NHNR 10  R 11 , --NHNHC(X 2 )NHR 12 , --NHSO 2  NR 8  R 9 , --NHNHC(O)R 12 , --NHNHSO 2  R 12  and --NHC(O)NR 8  R 9 . R 7  -R 12  are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl. X 1  and X 2  are selected independently from the group consisting of oxygen and sulfur. R 2  is hydrogen or halogen. R 3  -R 6  are selected independently from the group consisting of hydrogen, halogen, hydroxyl, --NR 8  R 9 , nitro, cyano, alkoxyl, lower alkyl, aryl and aryloxyl.

NOTICE OF U.S. GOVERNMENT RIGHTS

A portion of the work described herein was funded in part by SBIR GrantNo. 1 R43 CA65178-01. The U.S. Government may therefore have certainrights relating to this invention.

BACKGROUND OF THE INVENTION

1. The Field of the Invention

The present invention relates to human telomerase, a ribonucleoproteinenzyme involved in human telomere DNA synthesis, and to compounds thatinhibit telomerase activity. The invention provides methods, compoundsand compositions relating to the fields of molecular biology, chemistry,pharmacology, oncology and medicinal and diagnostic technology.

2. Description of Related Disclosures

The war on cancer has raged for over two decades. Yet, despite theexpenditure of over a billion dollars for research and development ofnew technologies to diagnose and treat malignancies, the age-adjustedcancer mortality rate in the U.S. has remained largely unchanged for thepast forty years. Indeed, if current epidemiological trends continue, itappears likely that cancer will overtake cardiovascular disease as theleading cause of death in the United States.

To be sure, some battles have been won and much has been learned aboutthe enemy. A few cancers (e.g., Hodgkin's disease) are now consideredcurable, and treatment regimes for many other cancers have improved overthe last decade. In addition, there has been an explosion of informationdescribing the regulatory mechanisms involved with the onset ofmalignancy, including the roles of growth factors, receptors, signaltransduction pathways, oncogenes, and tumor suppressor genes in thecontrol of cell growth and differentiation. However, these successes areovershadowed by the fact that cancer is a highly heterogeneous diseasein which profound differences exist in the mechanisms by which differentcell types become malignant. Thus, although we know more about themechanisms by which cells become malignant than ever before, each typeof cancer presents a unique set of problems in terms of treatment.

Because the cellular mechanisms leading to cancer are so heterogeneous,research on such mechanisms is unlikely to yield a general approach tocancer treatment that is effective and well tolerated by cancerpatients. Presently, a variety of non-specific treatment modalities areavailable, including surgery, radiation, and a variety of cytoreductiveand hormone-based drugs, used alone or in combination. Some oncolyticdrugs are also available, but the efficacy of these drugs varies amongcancer types. Thus, patients suffering from cancer often are forced toundergo treatments that are highly non-specific and highly toxic.Commonly, the toxicity of the treatments produces severe side effects,including nausea and vomiting, hair loss, diarrhea, fatigue, ulcerationsand the like, which severely impact the patient's quality of life. Insome cases, the impact on the patient's quality of life can be so greatthat the patient is unable to continue the full course of therapy oropts out of treatment entirely.

Recently, however, an understanding of the mechanisms by which normalcells reach the state of senescence, i.e., the loss of proliferativecapacity that cells normally undergo in the cellular aging process, hasbegun to emerge. The DNA at the ends, or telomeres, of the chromosomesof eukaryotes usually consists of tandemly repeated simple sequences.Scientists have long known that telomeres have an important biologicalrole in maintaining chromosome structure and function. More recently,scientists have speculated that the cumulative loss of telomeric DNAover repeated cell divisions may act as a trigger of cellular senescenceand aging, and that the regulation of telomerase, an enzyme involved inthe maintenance of telomere length, may have important biologicalimplications. See Harley, 1991, Mutation Research, 256:271-282,incorporated herein by reference.

Telomerase is a ribonucleoprotein enzyme that synthesizes one strand ofthe telomeric DNA using as a template a sequence contained within theRNA component of the enzyme. See Blackburn, 1992, Annu. Rev. Biochem,.61: 113-129, incorporated herein by reference. Methods for detectingtelomerase activity, as well as for identifying compounds that regulateor affect telomerase activity, together with methods for therapy anddiagnosis of cellular senescence and immortalization by controllingtelomere length and telomerase activity, have also been described. SeeKim, et al., 1994, Science, 266:2011-2014; PCT patent publication No.93/23572, published Nov. 25, 1993; U.S. patent application Ser.Nos.08/288,501, filed Aug. 10, 1994; 08/330,123, filed Oct. 27, 1994;08/272,102, filed Jul. 7, 1994; 08/255,774, filed Jun. 7, 1994;08/315,214 and 08/315,216, both of which were filed Sep. 28, 1994;08/151,477 and 08/153,051, both of which were filed 12 Nov. 1993;08/060,952, filed 13 May 1993; and 08/038,766, filed 24 Mar. 1993. Eachof the foregoing patent applications and reference is incorporatedherein by reference.

The identification of compounds that inhibit telomerase activityprovides important benefits to efforts at treating human disease.Compounds that inhibit telomerase activity can be used to treat cancer,as cancer cells express telomerase activity and normal human somaticcells do not express telomerase activity at biologically relevant levels(i.e., at levels sufficient to maintain telomere length over many celldivisions). Unfortunately, few such compounds have been identified andcharacterized. Hence, there remains a need for compounds that act astelomerase inhibitors and for compositions and methods for treatingcancer and other diseases in which telomerase activity is presentabnormally. The present invention meets these and other needs.

SUMMARY OF THE INVENTION

The present invention provides methods, compounds and compositions thatare highly unique, specific and effective for treating malignantconditions by targeting cells having telomerase activity. The methods,compounds and compositions of the invention can be applied to a widevariety of malignant cell types and avoid the problems inherent incurrent cancer treatment modalities, which are non-specific and toxic.

In a first aspect, the present invention provides methods andcompositions for treating cancer in which a therapeutically effectiveamount of a benzo b!thiophene derivative, having the structure shownbelow, in a pharmaceutically acceptable carrier, is administered to amammal: ##STR2## In the structure above, R₁ is selected from the groupconsisting of --OR₇, --NR₈ R₉, --NHNR₁₀ R₁₁, --NHNHC(X₂)NHR₁₂, --NHSO₂NR₈ R₉, --NHNHC(O)R₁₂, --NHNHSO₂ R₁₂ and --NHC(O)NR₈ R₉, where R₇ -R₁₂are selected independently from the group consisting of hydrogen, alkyl,aryl, aralkyl, heteroaryl and heteroaralkyl. X₁ and X₂ are selectedindependently from the group consisting of oxygen and sulfur. Further,in the above structure, R₂ is hydrogen or halogen, and R₃ -R₆ areselected independently from the group consisting of hydrogen, halogen,hydroxyl, --NR₈ R₉, nitro, cyano, alkoxyl, lower alkyl, aryl andaryloxyl.

Preferred benzo b!thiophene telomerase inhibitors include those in whichR₁ is --OR₇ (with R₇ being hydrogen or methyl) or --NHNH₂, R₂ or R₆ ishalogen or hydrogen and R₅ is alkyl or alkoxyl. Compounds in which R₂-R₄ are hydrogen, R₅ is methyl or methoxy and R₆ is chloro, arepreferred. Also preferred are compounds in which R₂ is chloro, R₃ and R₄are hydrogen, R₅ is methyl or methoxy and R₆ is hydrogen.

Other preferred benzo b!thiophene derivatives of the present inventionare those compounds characterized by X₁ being oxygen and R₁ (from theabove structure) being --NHNHC(X₂)NHR₁₂ where R₁₂ is aryl. As notedabove, X₂ may be oxygen or sulfur. Such compounds have the followingstructure: ##STR3## In this structure, R₁₃ -R₁₇ are selectedindependently from the group consisting of hydrogen, halogen, hydroxyl,nitro, cyano, amino, alkylamino, arylamino, dialkylamino, diarylamino,arylalkylamino, alkoxyl, alkyl, aryl and aryloxyl. Especially preferredderivatives are those in which X₂ is sulfur and R₁₃ -R₁₇ are selectedindependently from the group consisting of hydrogen and chloro.

In another aspect, the present invention provides novel methods,compositions and compounds relating to a class of telomerase inhibitingagents that are derivatives or analogues of fused, bicyclic pyridob!thiophenes, pyrido b!furans, or monocyclic pyridine ethers or pyridinethioethers. These telomerase inhibiting compounds have the generalstructure shown below: ##STR4## In this structure, X₃ is oxygen orsulfur; and the double dashed lines between X₄ and X₅ indicate anoptional double bond, which, when present, forms a fused, bicyclicpyrido b!furan or pyrido b!thiophene ring system, depending upon whetherX₃ is oxygen or sulfur, respectively. When the double bond is notpresent, the compound is a monocyclic pyridine ether or thioether, againdepending upon whether X₃ is oxygen or sulfur. X₄ is --CH₂ R₂₁ or --CR₂₁--, where R₂₁ is selected from the group consisting of aryl, heteroaryl,aralkyl, heteroaralkyl, alkylcarbonyl, arylcarbonyl,heteroalkylcarbonyl, heteroaralkylcarbonyl, aralkylcarbonyl,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl,carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl andarylsulfonyl; and X₅ is hydrogen, alkyl, hydroxyl, alkoxyl, aryloxyl,halogen, cyano, amino, alkylamino, arylamino, dialkylamino, diarylamino,arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl,carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl,arylsulfonyl or --CR₂₂ -- where R₂₂ is selected from the groupconsisting of hydrogen, amino, alkylamino, arylamino, dialkylamino,diarylamino, arylalkylamino, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl,arylalkylaminocarbonyl, hydroxyl, halogen, cyano, carboxyl,alkoxycarbonyl and aryloxycarbonyl. When X₄ is --CR₂₁ --, X₅ is --CR₂₂--, and X₄ and X₅ are joined by the above-mentioned double bond, to formthereby a fused, bicyclic pyrido b!thiophene (X₃ is sulfur) or pyridob!furan (X₃ is oxygen) ring system. When X₄ is --CH₂ R₂₁, X₅ is not--CR₂₂ --, thereby forming a pyridine ether (X₃ is oxygen) or thioether(X₃ is sulfur). R₁₈ and R₁₉ are selected independently from the groupconsisting of hydrogen, halogen, hydroxy, aryloxy, alkoxy, lower alkyl,aryl, heteroaryl, aralkyl and heteroaralkyl. R₂₀ is selected from thegroup consisting of alkoxymethyl, dialkoxymethyl, arylalkylaminomethyl,arylaminomethyl, alkylaminomethyl, aminomethyl, diarylaminomethyl,dialkylaminomethyl, hydroximinyl, iminyl, aldehyde, alkylcarbonyl,arylcarbonyl, alkyliminyl, aryliminyl, aralkyliminyl, alkoximinyl,aryloximinyl, heterocycleiminyl, alkoxycarbonyl, aryloxycarbonyl,carboxyl, alkene, --HC═NNHR₂₃ where R₂₃ is selected from the groupconsisting of hydrogen, alkyl, aryl, aralkyl, heterocycle,heterocyclealkyl, and --C(X₆)NHR₂₄ where X₆ is oxygen or sulfur and R₂₄is selected from the group consisting of hydrogen, aryl, arylsulfonyl,aralkyl, heterocycle and heterocyclealkyl.

One group of preferred compounds of this class include pyridob!thiophenes in which X₃ is sulfur, X₄ is --C(C(O)aryl)-- and X₅ is--C(NH₂)--, especially those pyrido b!thiophene compounds in which X₄ is--C(C(O)p-halophenyl)--.

Another group of compounds of this class includes monocyclic pyridineethers and thioethers having the following structure (X₄ is --CH₂ R₂₁):##STR5## In preferred compounds of the invention having this structure,X₃ is oxygen or sulfur; R₂₁ is selected from the group consisting ofaryl, heteroaryl, aralkyl and heteroaralkyl; X₅ is hydrogen or cyano;R₁₈ and R₁₉ are selected independently from the group consisting ofhydrogen, halogen, hydroxyl, lower alkyl, aryl, heteroaryl, aralkyl,alkoxy, aryloxy and heteroaralkyl; and R₂₀ is selected from the groupconsisting of alkoxymethyl, dialkoxymethyl, arylalkylaminomethyl,arylaminomethyl, alkylaminomethyl, aminomethyl, diarylaminomethyl,dialkylaminomethyl, hydroximinyl, iminyl, aldehyde, alkylcarbonyl,arylcarbonyl, alkyliminyl, aryliminyl, aralkyliminyl, alkoximinyl,aryloximinyl, heterocycleiminyl, alkoxycarbonyl, aryloxycarbonyl,carboxyl, alkene, --HC═NNHR₂₃ where R₂₃ is selected from the groupconsisting of hydrogen, alkyl, aryl, aralkyl, heterocycle,heterocyclealkyl, and --C(X₆)NHR₂₄ where X₆ is oxygen or sulfur and R₂₄is selected from the group consisting of hydrogen, aryl, arylsulfonyl,aralkyl, heterocycle and heterocyclealkyl.

Preferred pyridine thioethers include those in which X₃ is sulfur, X₅ iscyano and R₂₀ is --HC═NNHR₂₃, where R₂₃ is alkyl or heterocycle.

Yet other preferred pyridine thioether compounds of this invention havestructures in which R₂₀ is dimethoxymethyl or --CH═NNH--C(S)NH(aryl) andX₅ is cyano.

Another group of preferred pyridine thioethers includes compounds inwhich X₃ is sulfur, R₂₁ is aryl, X₅ is cyano, and R₂₀ is aryliminyl, asshown below: ##STR6## In such compounds, R₁₈ and R₁₉ are as specifiedfor the immediately preceding structure, and R₂₅ -R₃₄ are preferablyselected independently from the group consisting of hydrogen, halogen,lower alkyl, alkoxyl, nitro, cyano, alkylamino and dialkylamino.Further, R₃₁ and R₃₂ together may be points of attachment for the groups--C(O)--NR₃₅ C--(O)-- and --O--(CH₂)_(n) --O-- where n is 1 or 2 and R₃₅is hydrogen, aryl, aralkyl or alkyl.

Another preferred group of pyridine thioether compounds of the inventionhas the following structure: ##STR7## Again, R₁₈ and R₁₉ are as definedabove. In addition, R₃₆ -R₄₀ are selected independently from the groupconsisting of hydrogen, halogen, alkoxyl, hydroxyl, amino, alkylamino,dialkylamino, arylamino, diarylamino, arylalkylamino, cyano, carboxyl,alkoxycarbonyl, aryloxycarbonyl, aldehyde, arylcarbonyl, alkylcarbonyl,nitro and lower alkyl. Preferred compounds include those in which R₃₆-R₄₀ are selected independently from the group consisting of hydrogen,halogen and alkyl.

Still other preferred pyridine thioether compounds of this inventionhave structures in which R₂₀ is --CH═NNH(aryl) and X₅ is cyano, as shownbelow: ##STR8## In this structure, R₁₈ and R₁₉ again are as definedabove and R₄₁ -R₅₀ are selected independently from the group consistingof hydrogen, halogen, hydroxyl, amino, alkylamino, arylamino,dialkylamino, diarylamino, arylalkylamino, lower alkyl, alkoxyl, nitroand cyano.

The above benzo b!thiophene, pyrido b!thiophene, pyrido b!furan,pyridine ether and pyridine thioether derivatives have many valuableuses as inhibitors of deleterious telomerase activity, most importantly,the use to treat cancer in humans. The pharmaceutical compositions ofthis invention can be employed in treatment regimens in which cancercells are killed, in vivo, as demonstrated by the use of telomeraseinhibitors of the invention to kill cancer cells ex vivo. Thus, thisinvention provides therapeutic compositions for treating cancer, andmethods for treating cancer in humans and other mammals (e.g., cows,horses, sheep, steer, pigs and animals of veterinary interest such ascats and dogs).

These and other features of the invention will be described in detailbelow with reference to the associated structures and tables.

DESCRIPTION OF SPECIFIC EMBODIMENTS

I. Definitions

The term "alkyl" as used herein refers to a straight, branched or cyclichydrocarbon chain fragment or radical containing between about one andabout twenty carbon atoms, more preferably between about one and aboutten carbon atoms (e.g., methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, n-butyl, iso-butyl, tert-butyl, cyclobutyl, adamantyl,noradamantyl and the like). Straight, branched or cyclic hydrocarbonchains having eight or fewer carbon atoms will also be referred toherein as "lower alkyl". The hydrocarbon chains may further include oneor more degrees of unsaturation i.e., one or more double or triple bonds(e.g., vinyl, propargyl, allyl, 2-buten-1-yl, 2-cyclopenten-1-yl,1,3-cyclohexadien-1-yl, 3-cyclohexen-1-yl and the like). Alkyl groupscontaining double bonds such as just described will also be referred toherein as "alkenes". Similarly, alkyl groups having triple bonds willalso be referred to herein as "alkynes". However, as used in contextwith respect to cyclic alkyl groups, the combinations of double and/ortriple bonds do not include those bonding arrangements that render thecyclic hydrocarbon chain aromatic.

In addition, the term "alkyl" as used herein further includes one ormore substitutions at one or more carbon atoms of the hydrocarbonfragment or radical. Such substitutions include, but are not limited to:aryl; heterocycle; halogen; nitro (--NO₂); cyano (--CN); hydroxyl (alsoreferred to herein as "hydroxy"), alkoxyl (also referred herein asalkoxy) or aryloxyl (also referred to herein as "aryloxy", --OR); thioor mercapto, or alkyl or arylthioether (--SR); amino, alkylamino,arylamino, dialkyl- or diarylamino, or arylalkylamino (--NRR');aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl(--C(O)NRR'); carboxyl, or alkyl- or aryloxycarbonyl (--C(O)OR);aldehyde, or aryl- or alkylcarbonyl (RC(O)--); iminyl, or aryl- oralkyliminyl (--C(═NR)--); sulfo (--SO₂ OR); or alkyl- or arylsulfonyl(--SO₂ R); or hydroximinyl, or aryl- or alkoximinyl (--C(═NOR)--); whereR and R' independently are hydrogen, aryl or alkyl as defined herein.

The term "halogen" as used herein refers to the substituents fluoro,bromo, chloro, and iodo.

The term "carbonyl" as used herein refers to the functional group--C(O)--. However, it will be appreciated that this group may bereplaced with well-known groups that have similar electronic and/orsteric character, such as, but not limited to, sulfonyl (--SO₂ --),phosphonyl (--PO₂ --) and methylene (--C(CH₂)--). Other carbonylequivalents will be familiar to those having skill in the medicinal andorganic chemical arts.

The term "aryl" as used herein refers to cyclic aromatic carbon chainshaving twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyland anthracenyl. One or more carbon atoms may also be substituted with,e.g.: aryl; heterocycle; halogen; nitro (--NO₂); cyano (--CN); hydroxyl(also referred to herein as "hydroxy"), alkoxyl (also referred herein asalkoxy) or aryloxyl (also referred to herein as "aryloxy", --OR); thioor mercapto, or alkyl or arylthioether (--SR); amino, alkylamino,arylamino, dialkyl- or diarylamino, or arylalkylamino (--NRR');aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl(--C(O)NRR'); carboxyl, or alkyl- or aryloxycarbonyl (--C(O)OR);aldehyde, or aryl- or alkylcarbonyl (RC(O)--); iminyl, or aryl- oralkyliminyl (--C(═NR)--); sulfo (--SO₂ OR); or alkyl- or arylsulfonyl(--SO₂ R); or hydroximinyl, or aryl- or alkoximinyl (--C(═NOR)--); whereR and R' independently are hydrogen, aryl or alkyl as defined herein.

The term "aralkyl" as used herein refers to an aryl group that is joinedto a structure by one or more alkyl groups, e.g., benzyl,α-methylbenzyl, phenethyl, and the like.

The term "heterocycle" as used herein refers to a cyclic alkyl group oraryl group as defined above in which one or more carbon atoms of acyclic alkyl group has been replaced by a non-carbon atom, especiallynitrogen, oxygen or sulfur. Aromatic heterocycles are also referred toherein as "heteroaryl". For example, such groups include, but are notlimited to, furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl, thienyl,tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl,isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl,imidazolyl, imidazolinyl, pyridyl, pyridazinyl, triazinyl, piperidinyl,morpholinyl, thiomorpholinyl, pyrazinyl, piperazinyl, pyrimidinyl,naphthyridinyl, benzofuranyl, benzothienyl, indolyl, indolinyl,indolizinyl, indazolyl, quinolizinyl, quinolinyl, isoquinolinyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl,quinuclidinyl, carbazolyl, acridiniyl, phenazinyl, phenothiazinyl,phenoxazinyl, purinyl, benzimidazolyl and benzthiazolyl.

The above heterocyclic groups may further include one or moresubstituents at one or more carbon and/or non-carbon atoms of theheteroaryl group, e.g.: aryl; heterocycle; halogen; nitro (--NO₂); cyano(--CN); hydroxyl (also referred to herein as "hydroxy"), alkoxyl (alsoreferred herein as alkoxy) or aryloxyl (also referred to herein as"aryloxy", --OR); thio or mercapto, or alkyl or arylthioether (--SR);amino, alkylamino, arylamino, dialkyl- or diarylamino, or arylalkylamino(--NRR'); aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl(--C(O)NRR'); carboxyl, or alkyl- or aryloxycarbonyl (--C(O)OR);aldehyde, or aryl- or alkylcarbonyl (RC(O)--); iminyl, or aryl- oralkyliminyl (--C(═NR)--); sulfo (--SO₂ OR); or alkyl- or arylsulfonyl(--SO₂ R); or hydroximinyl, or aryl- or alkoximinyl (--C(═NOR)--); whereR and R' independently are hydrogen, aryl or alkyl as defined herein.

The term "heteroaralkyl" as used herein refers to a heteroaryl groupthat is joined to a parent structure by one or more alkyl groups, e.g.,2-thienylmethyl, and the like.

II. Telomerase Inhibitors

As noted above, the immortalization of cells involves inter alia theactivation of telomerase. More specifically, the connection betweentelomerase activity and the ability of many tumor cell lines, includingskin, connective tissue, adipose, breast, lung, stomach, pancreas,ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervoussystem (CNS), retina and blood tumor cell lines, to remain immortal hasbeen demonstrated by analysis of telomerase activity (Kim, et al.,incorporated herein by reference above). This analysis, supplemented bydata that indicates that the shortening of telomere length can providethe signal for replicative senescence in normal cells, see PCTApplication No. 93/23572 (incorporated herein by reference above),demonstrates that inhibition of telomerase activity can be an effectiveanti-cancer therapy. Thus, telomerase activity can prevent the onset ofotherwise normal replicative senescence by preventing the normalreduction of telomere length and the concurrent cessation of cellreplication that occurs in normal somatic cells after many celldivisions. In cancer cells, where the malignant phenotype is due to lossof cell cycle or growth controls or other genetic damage, an absence oftelomerase activity permits the loss of telomeric DNA during celldivision, resulting in chromosomal rearrangements and aberrations thatlead ultimately to cell death. However, in cancer cells havingtelomerase activity, telomeric DNA is not lost during cell division,thereby allowing the cancer cells to become immortal, leading to aterminal prognosis for the patient. Agents capable of inhibitingtelomerase activity in tumor cells offer therapeutic benefits withrespect to a wide variety of cancers and other conditions (e.g., fungalinfections) in which immortalized cells having telomerase activity are afactor in disease progression or in which inhibition of telomeraseactivity is desired for treatment purposes. The telomerase inhibitors ofthe invention can also be used to inhibit telomerase activity in germline cells, which may be useful for contraceptive purposes.

Thus, in one aspect, the present invention provides compounds that canserve as an important weapons against many types of malignancies in thewar against cancer. In particular, the compounds of the presentinvention can provide a highly general method of treating many--if notmost--malignancies, as demonstrated by the highly varied human tumorcell lines and tumors having telomerase activity. More importantly, thecompounds of the present invention can be effective in providingtreatments that discriminate between malignant and normal cells to ahigh degree, avoiding many of the deleterious side-effects present withmost current chemotherapeutic regimes which rely on agents that killdividing cells indiscriminately.

In one aspect, the present invention provides pharmaceuticalcompositions and methods relating to compounds having the generalstructure shown as Compound I below: ##STR9## In Compound I, R₁ isselected from the group consisting of --OR₇, --NR₈ R₉, --NHNR₁₀ R₁₁,--NHNHC(X₂)NHR₁₂, --NHSO₂ NR₈ R₉, --NHNHC(O)R₁₂, --NHNHSO₂ R₁₂ and--NHC(O)NR₈ R₉, where R₇ -R₁₂ are selected independently from the groupconsisting of hydrogen, alkyl, aryl, aralkyl, heteroaryl andheteroaralkyl. X₁ and X₂ are selected independently from the groupconsisting of oxygen and sulfur. Further, in the above structure, R₂ ishydrogen or halogen, and R₃ -R₆ are selected independently from thegroup consisting of hydrogen, halogen, hydroxyl, --NR₈ R₉, nitro, cyano,alkoxyl, lower alkyl, aryl and aryloxyl.

Certain of the benzo b!thiophene compounds of this invention includenovel compounds in which either or both R₂ or R₆ is halogen when R₇ ishydrogen, with the limitation that (i) R₂ is not chloro when: R₃, R₄ andR₆ are hydrogen, and R₅ is chloro, fluoro or hydrogen; or when R₃ and R₄are hydrogen, R₅ is methoxy and R₆ is chloro; or when R₃, R₅ and R₆ arehydrogen and R₄ is fluoro; or when R₃ is chloro and R₄ -R₆ are hydrogen;(ii) R₂ is not bromo when R₃ -R₆ are hydrogen, or when R₃ -R₅ arehydrogen and R₆ is methyl, or when R₃, R₅ and R₆ are hydrogen and R₄ ismethyl, chloro or methoxy, or when R₃ is chloro and R₄ -R₆ are hydrogen;and (iii) R₆ is not chloro when R₂ -R₅ are hydrogen.

Other novel compounds include those in which, when R₁ is --NHNR₁₀ R₁₁,R₁₁ is not tert-butyl, unsubstituted phenyl, hydrogen, 2-thienylmethyl,2-furanylmethyl, unsubstituted benzyl or α-methylbenzyl, or benzyl orα-methylbenzyl substituted with bromo, chloro, methoxy, methyl,dimethylamino, hydroxyl at the para position of the phenyl ring orsubstituted with --NH₂ or hydroxyl at the ortho position of the phenylring, when R₁₀ is hydrogen, R₂ is chloro and R₃ -R₆ are hydrogen. Inaddition, when R₁ is --NHNR₁₀ R₁₁, R₁₁ is not hydrogen when R₂ ischloro, R₃, R₄, R₆ and R₁₀ are hydrogen and R₅ is methoxy. Furthermore,R₁₁ is not tert-butyl when R₂, R₃, R₅, R₆ and R₁₀ are hydrogen and R₄ isfluoro, chloro, trifluoromethyl or hydrogen; or when R₂ -R₄, R₆ and R₁₀are hydrogen and R₅ is fluoro, chloro, methyl or trifluoromethyl; orwhen R₄ and R₅ are both methoxy, fluoro or chloro.

Still other novel compounds include those in which, when R₁ is--NHNHC(O)NHR₁₂, R₁₂ is not methyl, phenyl or phenyl substituted at thepara position with methoxy or chloro when R₂ is chloro, R₃, R₄ and R₆are hydrogen and R₅ is methoxy or hydrogen. In addition, when R₅ ishydrogen, R₁₂ also is not para-methylphenyl, unsubstituted benzyl orunsubstituted allyl.

In preferred embodiments, R₂ and R₆ are selected independently from thegroup consisting of hydrogen and halogen. Preferably at least one orboth of R₂ and R₆ is halogen, especially chloro. In one preferredembodiment of Compound I, X₁ is oxygen and R₁ is --OR₇. In anotherpreferred embodiment, X₁ is oxygen, R₁ is --OR₇ and R₇ is hydrogen ormethyl. Also preferred are compounds in which X₁ is oxygen, R₁ is --OH,--OCH₃ or --NHNH₂ and R₅ is lower alkyl, or alkoxyl. Other preferredembodiments of Compound I are those in which X₁ is oxygen, R₁ ishydroxyl or methoxy, R₂ and R₆ are hydrogen or chloro, R₃, and R₄ arehydrogen, and R₅ is methyl or methoxy. Some preferred compounds areshown below (Compounds II-IV). ##STR10##

Also preferred are compounds of the general formula shown in Compound Iin which R₁ is --NHNHC(X₂)NHR₁₂, where X₁ and X₂ are defined as abovewith respect to Compound I, R₂ is hydrogen or chloro, and R₁₂ is aryl.These compounds have the structure shown below as Compound V: ##STR11##In Compound V, R₁₃ -R₁₇ are selected independently from the groupconsisting of hydrogen, halogen, alkoxyl, lower alkyl, aryl andaryloxyl. As noted above, novel compounds include those in which R₁₅ isnot methoxy or chloro when R₂ is chloro, R₃, R₄, R₆, R₁₃, R₁₄, R₁₆ andR₁₇ are hydrogen and R₅ is methoxy or hydrogen. Also, when R₂ is chloro,R₃ -R₆, R₁₃, R₁₄, R₁₆ and R₁₇ are hydrogen, R₁₅ also is not methyl.

Preferred embodiments of Compound V include those in which X₁ is oxygenand X₂ is sulfur. Also preferred are those embodiments in which X₁ isoxygen, X₂ is sulfur and R₃ -R₆ and R₁₃ -R₁₇ are selected independentlyfrom the group consisting of hydrogen, halogen, hydroxyl, nitro, cyano,amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino,alkoxyl, alkyl, aryl and aryloxyl. Several preferred embodiments ofCompound V, in which X₁ is oxygen, X₂ is sulfur, R₂ is chloro, R₃ -R₆are hydrogen and R₁₃ -R₁₇ are hydrogen or chloro independently, areshown in Table 1 below (Compounds VI-X).

                  TABLE 1                                                         ______________________________________                                        Compound  R.sub.13 R.sub.14                                                                             R.sub.15                                                                              R.sub.16                                                                           R.sub.17                               ______________________________________                                        VI        H        Cl     H       H    Cl                                     VII       H        H      Cl      Cl   H                                      VIII      H        H      Cl      Cl   Cl                                     IX        H        Cl     Cl      H    Cl                                     X         H        H      Cl      H    Cl                                     ______________________________________                                    

In another embodiment, the present invention provides telomeraseinhibiting compounds having the structure shown as Compound XI, inaddition to related pharmaceutical compositions and therapeutic methodsand uses. ##STR12## In Compound XI, X₃ is oxygen or sulfur; and thedouble dashed lines indicate an optional double bond, which, whenpresent, forms a fused, bicyclic pyrido b!furan or pyrido b!thiophenering system, depending upon whether X₃ is oxygen or sulfur,respectively. When the double bond is not present, Compound XI is eithera pyridine ether or thioether, again depending upon whether X₃ is oxygenor sulfur. X₄ is --CH₂ R₂₁ or --CR₂₁ --, where R₂₁ is selected from thegroup consisting of aryl, heteroaryl, aralkyl, heteroaralkyl,alkylcarbonyl, arylcarbonyl, heteroalkylcarbonyl, heteroaralkylcarbonyl,aralkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl,carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl andarylsulfonyl. X₅ is hydrogen, alkyl, hydroxyl, alkoxyl, aryloxyl,halogen, cyano, amino, alkylamino, arylamino, dialkylamino, diarylamino,arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl,carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl,arylsulfonyl or --CR₂₂ -- where R₂₂ is selected from the groupconsisting of hydrogen, amino, alkylamino, arylamino, dialkylamino,diarylamino, arylalkylamino, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl,arylalkylaminocarbonyl, halogen, cyano, carboxyl, alkoxycarbonyl andaryloxycarbonyl. When X₄ is --CR₂₁ --, X₅ is --CR₂₂ --, and X₄ and X₅are joined by the above-mentioned double bond, to form thereby a fused,bicyclic pyrido b!thiophene (X₃ is sulfur) or pyrido b!furan (X₃ isoxygen) ring system. When X₄ is --CH₂ R₂₁, X₅ is not --CR₂₂ --, therebyforming a pyridine ether (X₃ is oxygen) or thioether (X₃ is sulfur).

In Compound XI above, R₁₈ and R₁₉ are selected independently from thegroup consisting of hydrogen, halogen, hydroxy, aryloxy, alkoxy, loweralkyl, aryl, heteroaryl, aralkyl and heteroaralkyl. R₂₀ is selected fromthe group consisting of alkoxymethyl, dialkoxymethyl,arylalkylaminomethyl, arylaminomethyl, alkylaminomethyl, aminomethyl,diarylaminomethyl, dialkylaminomethyl, hydroximinyl, iminyl, aldehyde,alkylcarbonyl, arylcarbonyl, alkyliminyl, aryliminyl, aralkyliminyl,alkoximinyl, aryloximinyl, heterocycleiminyl, alkoxycarbonyl,aryloxycarbonyl, carboxyl, alkene, --HC═NNHR₂₃ where R₂₃ is selectedfrom the group consisting of hydrogen, alkyl, aryl, aralkyl,heterocycle, heterocyclealkyl, and --C(X₆)NHR₂₄ where X₆ is oxygen orsulfur and R₂₄ is selected from the group consisting of hydrogen, aryl,arylsulfonyl, aralkyl, heterocycle and heterocyclealkyl.

Preferred telomerase inhibiting compounds having the formula shown inCompound XI include those in which X₃ is sulfur, R₂₀ is--HC═NNH(p-methylphenyl), X₄ is --CR₂₁ --, X₅ is --CR₂₂ -- and X₄ and X₅are joined by a double bond to form a pyrido b!thiophene ring system.Also preferred are those compounds in which R₂₁ is phenylcarbonyl andR₂₂ is amino. Also preferred are those compounds in which R₂₁ ispara-bromophenylcarbonyl or para-fluorophenylcarbonyl and R₂₂ is amino,as shown below (Compounds XII and XIII). Although only one stereoisomerfor each of Compound XII and XIII is shown, any structure shown hereinhaving more than one possible stereoisomer will be assumed to representalso each possible stereoisomer in pure form, in addition to a mixtureof stereoisomers unless otherwise indicated. Methods for isolatingand/or synthesizing pure stereoisomers can be performed using well knownmethods and materials. ##STR13##

Another preferred class of compounds related to Compound XI is that inwhich X₄ is --CH₂ R₂₁ and X₅ is cyano, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl orarylalkylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl or carboxyl,i.e., pyridine ethers and thioethers. This class is illustratedgenerally by Compound XIV below. ##STR14## Preferred embodiments ofCompound XIV are those in which X₃ is sulfur and X₅ is cyano. Alsopreferred are embodiments in which X₃ is sulfur, X₅ is cyano and R₂₀ isaryliminyl, heterocycleiminyl or alkyliminyl. Also preferred are thoseembodiments in which X₃ is sulfur, X₅ is cyano, R₂₀ is aryliminyl andR₂₁ is aryl or heteroaryl.

Especially preferred are those compounds in which R₂₁ is phenyl, asshown by Compound XV below: ##STR15## In Compound XV, R₂₅ -R₃₄ areselected independently from the group consisting of hydrogen, halogen,lower alkyl, alkoxyl, nitro, cyano, alkylamino and dialkylamino.Furthermore, R₃₁ and R₃₂ together may be points of attachment for thegroups --C(O)--NR₃₅ C--(O)-- and --O--(CH₂)_(n) --O-- where n is 1 or 2and R₃₅ is hydrogen, aryl, aralkyl or alkyl. Preferred compounds havingthe structure of Compound XV and the just-described substitution patternare those in which R₂₅ -R₂₉ are selected independently from the groupconsisting of hydrogen and halogen.

Especially preferred compounds (Compounds XVI-XXVII) of the classdefined by Compound XV are those in which R₁₈, R₁₉, R₂₅, R₂₆, R₂₉, R₃₀,R₃₃ and R₃₄ are hydrogen, and R₂₇, R₂₈, R₃₁ and R₃₂ are as shown belowin Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Compound   R.sub.27                                                                             R.sub.28   R.sub.31                                                                           R.sub.32                                    ______________________________________                                        XVI        Cl     H          H    OCH.sub.3                                   XVII       Cl     H          H    Cl                                          XVIII      Cl     H          H    CH.sub.3                                    XIX        Cl     Cl         H    OCH.sub.3                                   XX         Cl     Cl         H    Cl                                          XXI        Cl     Cl         H    CH.sub.3                                    XXII       Cl     Cl         CF.sub.3                                                                           Br                                          XXIII      Cl     Cl         NO.sub.2                                                                           Cl                                          XXIV       Cl     Cl         Cl   CN                                          XXV        Cl     Cl         F    F                                           XXVI       Cl     Cl         H    N(CH.sub.3).sub.2                           XXVII      Cl     Cl         Cl   Cl                                          ______________________________________                                    

Additional preferred compounds within the class shown generally byCompound XV above include compounds in which R₂₇, R₂₉, R₃₁ and R₃₂ arechloro and R₂₅, R₂₆, and R₂₈ are hydrogen (Compound XXVIII), andcompounds in which R₃₁ and R₃₂ are joined to form cyclic ethers andphthalimide derivatives (Compounds XXIX-XXXI) as shown below: ##STR16##

Still other preferred compounds include those having the generic formulaof Compound XV, but in which X₅ is cyano, R₂₁ is aryl and R₂₀ is alkyl-or heterocycleiminyl, and, more preferably, those compounds in which thealkyl substituent of the alkyliminyl group is cyclic alkyl. Alsopreferred are cyclic alkyl and heterocycle substituents having more thanabout six constituent atoms, such as Compounds XXXII-XXXVI below.##STR17##

The structure of each of compounds XVI-XXXIX is represented by thestructure of Compound XIV, in which R₂₀ is aryl-, alkyl- orheterocycleiminyl, R₂₁ is phenyl and X₃ is sulfur. Other preferredembodiments are those in which R₂₀ of Compound XIV above is an aldehyde(--C(O)H). Preferred compounds of this type include those in which X₅ iscyano. Also preferred are those in which X₅ is cyano and R₂₁ is aryl orheteroaryl. Especially preferred compounds have the structure shown forCompound XL below: ##STR18## In a preferred embodiment of Compound XL,R₃₆ -R₄₀ are selected independently from the group consisting ofhydrogen, halogen, alkoxyl, hydroxyl, amino, alkylamino, dialkylamino,arylamino, diarylamino, arylalkylamino, cyano, carboxyl, alkoxycarbonyl,aryloxycarbonyl, aldehyde, arylcarbonyl, alkylcarbonyl, nitro and loweralkyl. More preferred embodiments of Compound XL are those in which R₃₆R₃₇ and R₄₀ are hydrogen, R₃₈ is chloro and R₃₉ is hydrogen or chloro,Compounds XLI and XLII, are shown below: ##STR19##

Other preferred embodiments of Compound XIV are those in which R₂₀ isdimethoxymethyl (--CH(OCH₃)₂), and more preferably, those in which R₂₀is dimethoxymethyl and X₅ is cyano.

Another preferred embodiment of Compound XIV includes compounds in whichR₂₀ is --CH═NNHR₂₃, and, especially, those in which R₂₁ and R₂₃ are aryland X₅ is cyano. A preferred structure is shown below (Compound XLIII):##STR20## In Compound XLIII above, R₄₁ -R₅₀ are selected independentlyfrom the group consisting of hydrogen, halogen, hydroxyl, amino,alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, loweralkyl, alkoxyl, nitro and cyano. In a preferred embodiment of CompoundXLIII, R₄₁ -R₅₀ are selected independently from the group consisting ofhydrogen and halogen. Especially preferred are Compounds XLIV and XLVbelow: ##STR21##

In still another preferred embodiment of Compound XIV, R₂₀ is--HC═NNHR₂₃, where R₂₃ is --C(S)NHR₂₄ as described above. Preferably,R₂₄ is aryl. More preferably, R₂₁ and R₂₄ are aryl and X₅ is cyano asshown in Compound XLVI below. ##STR22## In Compound XLVI, R₅₁ -R₅₅ areselected independently from the group consisting of hydrogen, alkyl andhalogen, and R₅₆ -R₆₀ are selected independently from the groupconsisting of alkyl, aryl, heterocycle, halogen, nitro, cyano, hydroxyl,alkoxyl or aryloxyl, amino, alkylamino, arylamino, dialkylamino,diarylamino, arylalkylamino, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, dialkylaminocarbonyl or arylalkylaminocarbonyl,carboxyl, alkoxycarbonyl, aryloxycarbonyl, aldehyde, or arylcarbonyl,alkylcarbonyl, iminyl, aryliminyl, alkyliminyl, sulfo, alkylsulfonyl,arylsulfonyl, hydroximinyl, aryloximinyl and alkoximinyl. More preferredembodiments of Compound XLVI are Compounds XLVII and XLVIII below.##STR23## III. Synthesis of Telomerase Inhibitors

The compounds of the present invention can be synthesized usingtechniques and materials known to those of skill in the art, such asdescribed, for example, in March, ADVANCED ORGANIC CHEMISTRY 4^(th) Ed.,(Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 3^(rd) Ed.,Vols. A and B (Plenum 1992), and Green and Wuts, PROTECTIVE GROUPS INORGANIC SYNTHESIS 2^(nd) Ed. (Wiley 1991), each of which is incorporatedherein by reference. Starting materials for the compounds of theinvention may be obtained using standard techniques and commerciallyavailable precursor materials, such as those available from AldrichChemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.),Lancaster Synthesis (Windham, N.H.), Apin Chemicals, Ltd. (NewBrunswick, N.J.), Ryan Scientific (Columbia, S.C.), Maybridge (Cornwall,England) and Trans World Chemicals (Rockville, Md.).

The procedures described herein for synthesizing the compounds of theinvention may include one or more steps of protection and deprotection(e.g., the formation and removal of acetal groups). In addition, thesynthetic procedures disclosed below can include various purifications,such as column chromatography, flash chromatography, thin-layerchromatography (TLC), recrystallization, distillation, high-pressureliquid chromatography (HPLC) and the like. Also, various techniques wellknown in the chemical arts for the identification and quantification ofchemical reaction products, such as proton and carbon-13 nuclearmagnetic resonance (¹ H and ¹³ C NMR), infrared and ultravioletspectroscopy (IR and UV), X-ray crystallography, elemental analysis(EA), HPLC and mass spectroscopy (MS) can be used as well. Methods ofprotection and deprotection, purification and identification andquantification are well known in the chemical arts.

Compounds of the class represented by Compound I can be synthesizedusing the following general procedure (Scheme 1). ##STR24##

Starting from the appropriately substituted cinnamic acid 1, preparedfrom standard methods and procedures or purchased commercially (e.g.,Aldrich), reaction with thionyl chloride under reflux provides thecorresponding 3-chlorobenzo b!thiophene-2-carbonyl chloride 2. Reactionof the acid chloride 2 using, e.g., trimethylsilyl chloride (Si(CH₃)₃Cl) and an alcohol (e.g., methanol) provides the corresponding ester 3.Ester 3 corresponds generally to Compounds II-IV above. If the hydrazideis desired, that derivative can be prepared from the ester 3 by reactionwith hydrazine in methanol to produce the hydrazide 4. Additionalhydrazide derivatives, containing carbonyl or thiocarbonyl substituentson the terminal hydrazide nitrogen, can be prepared, e.g., by reactionof hydrazide 4 with an appropriately substituted isocyanate orisothiocyanate 5 to produce the corresponding addition product 6. Thisaddition product corresponds to Compounds V-X, illustrated above.Examples of each of the transformations outlined above are presentedbelow. The above-described transformations are well known in the art(see, e.g., Reid, W., et al. 1980. Liebigs. Ann. Chem. 1424-1427;Connor, D. T., et al. 1992. J. Med. Chem. 35(5):958-965; Higa, T., andKrubsack, A. J. 1976. J. Org. Chem. 41(21): 3399-3403; and Higa, T., andKrubsack, A. J. 1976. J. Org. Chem. 40(21): 3037-3045, each which isincorporated herein by reference.)

Compounds of the class represented by Compound XI can be synthesizedusing the following general procedure (Scheme 2). The use of thesubstituent identifiers R, R', R" and R'" is merely to indicate thepresence of one or more substituents at the position or moietyindicated. The values of R, R', R" and R'" shown in Scheme 2 below canbe determined by reference to the specific moieties described above inconnection with the structure of Compound XI. ##STR25##

With the appropriately substituted derivative of commercially available2-thio-3-cyano-5-(dimethoxymethyl)pyridine 7, reaction with anappropriate alkylating agent or other nucleophile under basic conditions(e.g., potassium carbonate (K₂ CO₃) in dimethylformamide (DMF) followedby addition of R'X, where X is a halide or other suitable leaving groupsuch as tosyl) provides the desired thioether 8. Removal of the acetalgroup using standard conditions (e.g., p-toluenesulfonic acid inbenzene), provides aldehyde 9 (pathway A).

Formation of imine 10 can be accomplished by conversion of the acetal 8or aldehyde 9 by reaction with an appropriately substituted amine understandard conditions, e.g., by heating the acetal or aldehyde with theamine in benzene with 3 Å molecular sieves in the presence of an acid(pathways B and D). Similarly, reaction of the acetal 8 with anappropriately substituted hydrazide or semicarbazide and acid (e.g.,HCl), provides the desired hydrazide 11 (pathway C). Alternatively, insome cases the imine 10 can be formed directly from the correspondingaldehyde 9 (Pathway D). Additional synthetic routes to compounds 9, 10,and 11 will be apparent to those having skill in the art based on thedisclosure herein.

A more specific example of the general synthetic procedure justdescribed above is presented below (Scheme 3). Again, R, R' and R" areas defined above. ##STR26##

Starting from commercially available2-thio-3-cyano-5-(dimethoxymethyl)pyridine 7 (Ryan Scientific), removalof the thiol proton using base (B:, e.g., K₂ CO₃ in DMF) andintroduction of the appropriately substituted benzylchloride derivative12 under substantially anhydrous conditions provides the benzylthioether13. Removal of the dimethylacetal group under acidic conditions,followed by imine formation with aniline derivative 14 produces thedesired arylimine 15. The benzyl chloride and aniline derivatives can bepurchased commercially (e.g., Aldrich) or made using standard methods.The reactions to form the thioether 13 and the imine 15 are performedusing standard methods.

Protocols from which the individual Compounds II-IV, VI-X, XII, XIII,XVI-XXXIX, XLI, XLII, XLIV, XLV, XLVII and XLVIII above can besynthesized are provided in the Examples below.

IV. Anti-Tumor Activity of the Telomerase Inhibitors of the Invention

The compounds of the present invention demonstrate inhibitory activityagainst telomerase in vitro and in vivo, as has been and can bedemonstrated as described below. As used herein, the term "in vitro"refers to tests performed using living cells in tissue culture. Suchprocedures are also known as "ex vivo".

One method used to identify compounds of the invention that inhibittelomerase activity involves placing cells, tissues, or preferably acellular extract or other preparation containing telomerase in contactwith several known concentrations of a test compound in a buffercompatible with telomerase activity. The level of telomerase activityfor each concentration of test compound is measured and the IC₅₀ (theconcentration of the test compound at which the observed activity for asample preparation was observed to fall one-half of its original or acontrol value) for the compound is determined using standard techniques.Other methods for determining the inhibitory concentration of a compoundof the invention against telomerase can be employed as will be apparentto those of skill in the art based on the disclosure herein.

A particularly preferred version of this method includes the followingsteps: (1) incubating a compound of the present invention at a knownconcentration (e.g., 1000 μM, 320 μM, 100 μM, 32 μM, 10 μM, 3.2 μM and1.0 μM in DMSO) with telomerase in the presence of an oligonucleotidesubstrate for human telomerase in a reaction mixture comprisingdeoxyribonucleotide triphosphates and a buffer in which telomerase isactive; (2) immobilizing non-chromosomal DNA (either together with, orseparated from, chromosomal DNA) from the reaction mixture resultingfrom step (1) on a solid support; (3) hybridizing to the DNA a labeledoligonucleotide probe complementary to a nucleic acid sequence added tothe substrate by telomerase; and (4) measuring an amount of the labeledprobe specifically hybridized to the DNA in comparison to a controlreaction in which no test compound is present. A detailed description ofappropriate materials, conditions, procedures, etc. for carrying outeach step of this method is provided in U.S. patent application Ser. No.08/288,501, filed Aug. 10, 1994 (previously incorporated herein byreference).

With the above-described methods, IC₅₀ values for several of thecompounds of the present invention were determined. The values reportedin Table 3 below are only approximate values; more exact IC₅₀ values canbe obtained by repetitive testing.

                  TABLE 3                                                         ______________________________________                                        Compound  IC.sub.50 (μM)                                                                         Compound  IC.sub.50 (μM)                             ______________________________________                                        II        7           XXVI      29                                            III       2           XXVII     29                                            IV        3           XXVIII    29                                            VI        24          XXIX      10                                            VII       12          XXX       10                                            VIII      11          XXXI      13                                            IX        12          XXXII     15                                            X         15          XXXIII    15                                            XII       20          XXXIV     8                                             XIII      20          XXXVI     18                                            XVI       29          XXXVII    4                                             XVII      29          XXXVIII   16                                            XVIII     29          XXXIX     13                                            XIX       7           XLI       10                                            XX        12          XLII      6                                             XXI       9           XLIV      19                                            XXII      10          XLV       19                                            XXIII     20          XLVII     18                                            XXIV      13          XLVIII    18                                            XXV       13                                                                  ______________________________________                                    

As shown in Table I, all of the compounds have anti-telomerase activity(i.e., IC₅₀ <50 μM). Several of the compounds of the invention (II-IV,VII-X, XIX-XXII, XXIV, XXV, XXIX-XXXIV, XXXIX, XLI, and XLII) are potenttelomerase inhibitors, having approximate IC₅₀ values of less than about15 μM.

Compounds of the invention also demonstrate anti-telomerase activity invitro. In one example, Compound XVIII was found to induce crisis inhuman immortal embryonic kidney cells in which telomerase activity waspresent. At a concentration of 3.2 μM, this compound induced crisis inHEK-293 cells within 16 weeks, and caused a reduction of telomere lengthby about 330 base pairs (bp). At a concentration of 10 μM, crisis wasinduced within 12 weeks and caused a 350 bp reduction in telomerelength.

In another example, Compound XVIII induced telomere reduction duringcell division in human ovarian tumor cell lines (OVCAR-5 and SK-OV-3) ata concentration of 10 μM. Importantly, however, in normal BJ cells offibroblast origin (used as a control), the observed reduction intelomere length was found to be no different from cells treated with adimethyl sulfoxide (DMSO) control. Compound XVIII also demonstrated nosignificant cytotoxic effects at a concentration below about 15 μM inthe same cells, although some delay through mitosis was observed by cellcycle analysis at concentrations between 5 μM and 15 μM.

In addition, Compound XVIII was found to be highly specific fortelomerase in comparison to other enzymes in vitro, as demonstrated by acomparison of the IC₅₀ for Compound XVIII against telomerase, andproteins having similar nucleic acid binding or modifying activitysimilar to telomerase, including DNA Polymerase I, HeLa RNA PolymeraseII, T3 RNA Polymerase, MMLV Reverse Transcriptase, Topoisomerase I,Terminal Transferase and Single-Stranded DNA Binding Protein (SSB).Compound XVIII was also found to be highly specific for telomerase ascompared to Glucose-6-Phosphate Dehydrogenase. These tests showed thatCompound XVIII was highly specific for telomerase at concentrationseffective in in vitro testing.

In vivo testing can also be performed using a mouse xenograft model inwhich OVCAR-5 tumor cells are grafted onto nude mice. As discussed inExample B. 2 below, mice treated with Compound XVIII are expected tohave tumor masses that, on average, may increase for a period followingthe initial dosing, but will begin to shrink in mass with continuingtreatment. In contrast, mice treated with a control (DMSO) are expectedto have tumor masses that continue to increase. Compound XVIII of theinvention was found to be highly effective in halting the growth oftumor cells in vitro and is expected to be highly effective in vivo.

From the foregoing those skilled in the art will appreciate that thepresent invention also provides methods for selecting treatment regimensinvolving administration of a compound of the invention. For suchpurposes, it may be helpful to perform a TRF analysis in which DNA fromtumor cells is analyzed by digestion with restriction enzymes specificfor sequences other than the telomeric (T₂ AG₃)_(N) sequence. Followingdigestion of the DNA, gel electrophoresis is performed to separate therestriction fragments according to size. The separated fragments arethen probed with nucleic acid probes specific for telomeric sequences todetermine the lengths of the telomeres of the cells in the sample. Bymeasuring the length of telomeric DNA, one can estimate how long atelomerase inhibitor should be administered and whether other methods oftherapy (e.g., surgery, chemotherapy and/or radiation) should also beemployed. In addition, during treatment, one can test cells to determinewhether a decrease in telomere length over progressive cell divisions isoccurring to demonstrate treatment efficacy.

V. Telomerase Inhibiting Compositions and Methods for Treating DiseasesWith the Same

The present invention also provides pharmaceutical compositions fortreating cancer and other conditions in which inhibition of telomeraseis an effective therapy. These compositions include a therapeuticallyeffective amount of a telomerase inhibiting compound of the invention ina pharmaceutically acceptable carrier or salt.

In one preferred embodiment, the present invention provides acomposition effective for treating cancer in a mammal. The compositionincludes a therapeutically effective amount of a compound having thestructure shown below in a pharmaceutically acceptable carrier:##STR27## In the structure, R₁ is selected from the group consisting of--OR₇, --NR₈ R₉, --NHNR₁₀ R₁₁, --NHNHC(X₂)NHR₁₂, --NHSO₂ NR₈ R₉,--NHNHC(O)R₁₂, --NHNHSO₂ R₁₂ and --NHC(O)NR₈ R₉, where R₇ -R₁₂ areselected independently from the group consisting of hydrogen, alkyl,aryl, aralkyl, heteroaryl and heteroaralkyl. X₁ and X₂ are selectedindependently from the group consisting of oxygen and sulfur. Further,in the above structure, R₂ is hydrogen or halogen, and R₃ -R₆ areselected independently from the group consisting of hydrogen, halogen,hydroxyl, --NR₈ R₉, nitro, cyano, alkoxyl, lower alkyl, aryl andaryloxyl.

In another aspect, the present invention includes a composition fortreating cancer in a mammal, comprising a therapeutically effectiveamount of any of the compounds embraced by the structure of Compound XIin a pharmaceutically acceptable carrier.

In addition, it will be appreciated that therapeutic benefits can berealized by combining a telomerase inhibitor of the invention with otheranti-cancer agents (including other inhibitors of telomerase) fortreatment of cancer. The choice of such combinations will depend onvarious factors including, but not limited to, the type of disease, theage and general health of the patient, the aggressiveness of diseaseprogression, the TRF length and telomerase activity of the diseasedcells to be treated and the ability of the patient to tolerate theagents that comprise the combination. For example, in cases where tumorprogression has reached an advanced state, it may be advisable tocombine a telomerase inhibiting compound of the invention with otheragents and therapeutic regimens that are effective at reducing tumorsize (e.g. radiation, surgery, chemotherapy and/or hormonal treatments).In addition, in some cases it may be advisable to combine a telomeraseinhibiting agent of the invention with one or more agents that treat theside effects of a disease, e.g., an analgesic, or agents effective tostimulate the patient's own immune response (e.g., colony stimulatingfactor).

In one such method, a pharmaceutical formulation comprises a telomeraseinhibitor of the invention with an anti-angiogenesis agent, such asfumagillin, fumagillin derivatives, or AGM-1470. The latter compound isavailable from Takeda Chemical Industries, Ltd., while the formercompounds are described in Ingber, et al., 6 Dec. 1990, "Syntheticanalogues of fumagillin that inhibit angiogenesis and suppress tumorgrowth", Nature 348:555-557, incorporated herein by reference for allpurposes. Other combinations may include, but are not limited to, atelomerase inhibitor of the invention in addition to one or moreantineoplastic agents or adjuncts (e.g., folinic acid or mesna).

Antineoplastic agents suitable for combination with the compounds of thepresent invention include, but are not limited to, alkylating agentsincluding alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines, such as a benzodizepa, carboquone, meturedepa and uredepa;ethylenimines and methylmelamines such as altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; nitroso ureas, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine and ranimustine. Additional agentsinclude dacarbazine, mannomustine, mitobronitol, mitolactol andpipobroman. Still other classes of relevant agents include antibiotics,hormonal antineoplastics and antimetabolites. Yet other combinationswill be apparent to those of skill in the art.

Additional agents suitable for combination with the compounds of thepresent invention include protein synthesis inhibitors such as abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, andO-methyl threonine. Additional protein synthesis inhibitors includemodeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,ricin, α-sarcin, shiga toxin, showdomycin, sparsomycin, spectinomycin,streptomycin, tetracycline, thiostrepton and trimethoprim. Inhibitors ofDNA synthesis, including alkylating agents such as dimethyl sulfate,mitomycin C, nitrogen and sulfur mustards, MNNG and NMS; intercalatingagents such as acridine dyes, actinomycins, adriamycin, anthracenes,benzopyrene, ethidium bromide, propidium diiodide intertwining agentssuch as distamycin and netropsin, can also be combined with compounds ofthe present invention in pharmaceutical compositions. DNA base analogssuch as acyclovir, adenine β-1-D-arabinoside, amethopterin, aminopterin,2-aminopurine, aphidicolin, 8-azaguanine, azaserine, 6-azauracil,2'-azido-2'-deoxynucleosides, 5-bromodeoxycytidine, cytosineβ-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, 5-fluorouracil, hydroxyureaand 6-mercaptopurine also can be used in combination therapies with thecompounds of the invention. Topoisomerase inhibitors, such ascoumermycin, nalidixic acid, novobiocin and oxolinic acid, inhibitors ofcell division, including colcemide, colchicine, vinblastine andvincristine, and RNA synthesis inhibitors including actinomycin D,α-amanitine and other fungal amatoxins, cordycepin (3'-deoxyadenosine),dichlororibofuranosyl benzimidazole, rifampicine and streptovaricin andstreptolydigin also can be combined with the compounds of the inventionto provide pharmaceutical compositions.

In another embodiment, the present invention includes compounds andcompositions in which a telomerase inhibitor is either combined with orcovalently bound to a cytotoxic agent bound to a targeting agent, suchas a monoclonal antibody (e.g., a murine or humanized monoclonalantibody). It will be appreciated that the latter combination may allowthe introduction of cytotoxic agents into cancer cells with greaterspecificity. Thus, the active form of the cytotoxic agent (i.e., thefree form) will be present only in cells targeted by the antibody. Ofcourse, the telomerase inhibitors of the invention may also be combinedwith monoclonal antibodies that have therapeutic activity againstcancer.

In general, a suitable effective dose of a compound of the inventionwill be in the range of 0.001 to 1000 milligram (mg) per kilogram (kg)of body weight of the recipient per day, preferably in the range of0.001 to 100 mg per kg of body weight per day, more preferably betweenabout 0.1 and 100 mg per kg of body weight per day and still morepreferably in the range of between 0.1 to 10 mg per kg of body weightper day. The desired dosage is preferably presented in one, two, three,four, or more subdoses administered at appropriate intervals throughoutthe day. These subdoses can be administered as unit dosage form, forexample, containing 5 to 10,000 mg, preferably 10 to 1000 mg of activeingredient per unit dosage from. Preferably, the dosage is presentedonce per day at a dosing at least equal to TID.

The composition used in these therapies can be in a variety of forms.These include, for example, solid, semi-solid, and liquid dosage forms,such as tablets, pills, powders, liquid solutions or suspensions,liposomes, and injectable and infusible solutions. The preferred formdepends on the intended mode of administration and therapeuticapplication. The compositions also preferably include conventionalpharmaceutically acceptable carriers and adjuvants, as is well known tothose of skill in the art. See, e.g., REMINGTON'S PHARMACEUTICALSCIENCES, Mack Publishing Co.: Easton, Pa., 17th Ed. (1985). Preferably,administration will be by oral or parenteral (including subcutaneous,intramuscular, intravenous, and intradermal) routes. More preferably,the route of administration will be oral. The therapeutic methods andagents of this invention can of course be used concomitantly or incombination with other methods and agents for treating a particulardisease or disease condition.

While it is possible to administer the active ingredient of thisinvention alone, it is preferable to present a therapeutic agent as partof a pharmaceutical formulation or composition. The formulations of thepresent invention comprise at least one telomerase activity-inhibitingcompound of this invention in a therapeutically or pharmaceuticallyeffective dose together with one or more pharmaceutically ortherapeutically acceptable carriers and optionally other therapeuticingredients. Various considerations for preparing such formulations aredescribed, e.g., in Gilman et al. (eds.) GOODMAN AND GILMAN'S: THEPHARMACOLOGICAL BASES OF THERAPEUTICS, 8th Ed., Pergamon Press (1990);and REMINGTON'S supra, each of which is incorporated herein by referencefor all purposes. Methods for administration are discussed therein,e.g., for oral, intravenous, intraperitoneal, intramuscular, and otherforms of administration. Generally, oral administration is preferred.

Typically, methods for administering pharmaceutical compositions will beeither topical, parenteral, or oral administration methods forprophylactic and/or therapeutic treatment. Oral administration is apreferred. The pharmaceutical compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. As noted above, unit dosage forms suitable for oraladministration include powders, tablets, pills, and capsules.

One can use topical administration to deliver a compound of theinvention by percutaneous passage of the drug into the systemiccirculation of the patient. The skin sites include anatomic regions fortransdermally administering the drug, such as the forearm, abdomen,chest, back, buttock, and mastoidal area. The compound is administeredto the skin by placing on the skin either a topical formulationcomprising the compound or a transdermal drug delivery device thatadministers the compound. In either embodiment, the delivery vehicle isdesigned, shaped, sized, and adapted for easy placement and comfortableretention on the skin.

A variety of transdermal drug delivery devices can be employed with thecompounds of this invention. For example, a simple adhesive patchcomprising a backing material and an acrylate adhesive can be prepared.The drug and any penetration enhancer can be formulated into theadhesive casting solution. The adhesive casting solution can be castdirectly onto the backing material or can be applied to the skin to forman adherent coating. See, e.g., U.S. Pat. Nos. 4,310,509; 4,560,555; and4,542,012.

In other embodiments, the compound of the invention will be deliveredusing a liquid reservoir system drug delivery device. These systemstypically comprise a backing material, a membrane, an acrylate basedadhesive, and a release liner. The membrane is sealed to the backing toform a reservoir. The drug or compound and any vehicles, enhancers,stabilizers, gelling agents, and the like are then incorporated into thereservoir. See, e.g., U.S. Pat. Nos. 4,597,961; 4,485,097; 4,608,249;4,505,891; 3,843,480; 3,948,254; 3,948,262; 3,053,255; and 3,993,073.

Matrix patches comprising a backing, a drug/penetration enhancer matrix,a membrane, and an adhesive can also be employed to deliver a compoundof the invention transdermally. The matrix material typically willcomprise a polyurethane foam. The drug, any enhancers, vehicles,stabilizers, and the like are combined with the foam precursors. Thefoam is allowed to cure to produce a tacky, elastomeric matrix which canbe directly affixed to the backing material. See, e.g., U.S. Pat. Nos.4,542,013; 4,460,562; 4,466,953; 4,482,534; and 4,533,540.

Also included within the invention are preparations for topicalapplication to the skin comprising a compound of the invention,typically in concentrations in the range from about 0.001% to 10%,together with a non-toxic, pharmaceutically acceptable topical carrier.These topical preparations can be prepared by combining an activeingredient according to this invention with conventional pharmaceuticaldiluents and carriers commonly used in topical dry, liquid, and creamformulations. Ointment and creams may, for example, be formulated withan aqueous or oily base with the addition of suitable thickening and/orgelling agents. Such bases may include water and/or an oil, such asliquid paraffin or a vegetable oil, such as peanut oil or castor oil.Thickening agents that may be used according to the nature of the baseinclude soft paraffin, aluminum stearate, cetostearyl alcohol, propyleneglycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax,and the like.

Lotions may be formulated with an aqueous or oily base and will, ingeneral, also include one or more of the following: stabilizing agents,emulsifying agents, dispersing agents, suspending agents, thickeningagents, coloring agents, perfumes, and the like. Powders may be formedwith the aid of any suitable powder base, e.g., talc, lactose, starch,and the like. Drops may be formulated with an aqueous base ornon-aqueous base also comprising one or more dispersing agents,suspending agents, solubilizing agents, and the like. Topicaladministration of compounds of the invention may also be preferred fortreating diseases such as skin cancer and fungal infections of the skin(pathogenic fungi typically express telomerase activity).

The topical pharmaceutical compositions according to this invention mayalso include one or more preservatives or bacteriostatic agents, e.g.,methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol,benzalkonium chlorides, and the like. The topical pharmaceuticalcompositions also can contain other active ingredients such asantimicrobial agents, particularly antibiotics, anesthetics, analgesics,and antipruritic agents.

The compounds of the present invention can also be delivered throughmucosal membranes. Transmucosal (i.e., sublingual, buccal, and vaginal)drug delivery provides for an efficient entry of active substances tosystemic circulation and reduces immediate metabolism by the liver andintestinal wall flora. Transmucosal drug dosage forms (e.g., tablet,suppository, ointment, pessary, membrane, and powder) are typically heldin contact with the mucosal membrane and disintegrate and/or dissolverapidly to allow immediate systemic absorption. Note that certain suchroutes may be used even where the patient is unable to ingest atreatment composition orally. Note also that where delivery of atelomerase inhibitor of the invention would be enhanced, one can selecta composition for delivery to a mucosal membrane, e.g., in cases ofcolon cancer one can use a suppository to deliver the telomeraseinhibitor.

For delivery to the buccal or sublingual membranes, typically an oralformulation, such as a lozenge, tablet, or capsule, will be used. Themethod of manufacture of these formulations is known in the art,including, but not limited to, the addition of the pharmacological agentto a pre-manufactured tablet; cold compression of an inert filler, abinder, and either a pharmacological agent or a substance containing theagent (as described in U.S. Pat. No. 4,806,356); and encapsulation.Another oral formulation is one that can be applied with an adhesive,such as the cellulose derivative hydroxypropyl cellulose, to the oralmucosa, for example as described in U.S. Pat. No. 4,940,587. This buccaladhesive formulation, when applied to the buccal mucosa, allows forcontrolled release of the pharmacological agent into the mouth andthrough the buccal mucosa.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly, or intravenously. Thus, thisinvention provides compositions for intravenous administration thatcomprise a solution of a compound of the invention dissolved orsuspended in an acceptable carrier. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, bufferedwater, saline, dextrose, glycerol, ethanol, or the like. Thesecompositions will be sterilized by conventional, well knownsterilization techniques, such as sterile filtration. The resultingsolutions can be packaged for use as is or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. In addition, if desired, the pharmaceutical compositionsto be administered may also contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agentsand the like, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate, etc. Such formulations will be useful intreating ovarian cancers.

Another method of parenteral administration employs the implantation ofa slow-release or sustained-release system, such that a constant levelof dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795,incorporated herein by reference.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc., an active compound as definedabove and optional pharmaceutical adjuvants in an excipient, such as,for example, water, saline, aqueous dextrose, glycerol, ethanol, oliveoil, and other lipophilic solvents, and the like, to form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,and the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known and will be apparent tothose skilled in this art; for example, see REMINGTON'S PHARMACEUTICALSCIENCES, supra. The composition or formulation to be administered willcontain an effective amount of an active compound of the invention.

For solid compositions, conventional nontoxic solid carriers can be usedand include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 0.1-95% of activeingredient, preferably about 20%.

The compositions containing the compounds of the invention can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a patientalready suffering from a disease, as described above, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease and its complications. An amount adequate to accomplish this isdefined as a "therapeutically effective amount or dose." Amountseffective for this use will depend on the severity of the disease andthe weight and general state of the patient.

In addition to internal (in vivo) administration, the compounds andcompositions of the invention may be applied ex vivo to achievetherapeutic effects, as for example, in the case of a patient sufferingfrom leukemia. In such an application, cells to be treated, e.g., bloodor bone marrow cells, are removed from a patient and treated with apharmaceutically effective amount of a compound of the invention. Thecells are returned to the patient following treatment. Such a procedurecan allow for exposure of cells to concentrations of therapeutic agentfor longer periods or at higher concentrations than otherwise available.

Once improvement of the patient's conditions has occurred, as, forexample, by the occurrence of remission in the case of a cancer patient,a maintenance dose is administered, if necessary. Subsequently, thedosage or the frequency of administration, or both, can be reduced, as afunction of the systems, to a level at which the improved condition isretained. When the symptoms have been alleviated to the desired level,treatment can cease. Patients can, however, require additional treatmentupon any recurrence of the disease symptoms.

In prophylactic applications (e.g. chemoprevention), compositionscontaining the compounds of the invention are administered to a patientsusceptible to or otherwise at risk of a particular disease. Such anamount is defined to be a "prophylactically effective amount or dose."In this use, the precise amounts again depend on the patient's state ofhealth and weight.

As will be apparent to those of skill in the art upon reading of thisdisclosure, the present invention provides valuable reagents relating tohuman telomerase. The above description of necessity provides a limitedand merely illustrative sampling of specific compounds, and should notbe construed as limiting the scope of the invention. Other features andadvantages of the invention will be apparent from the following examplesand claims.

EXAMPLES

The following examples describe specific aspects of the invention toillustrate the invention and also provide a description of the methodsused to identify and test compounds that inhibit the activity oftelomerase to aid those of skill in the art in understanding andpracticing the invention. The examples should not be construed aslimiting the invention, in any manner.

A. Chemical Syntheses

1. Synthesis of Compounds I-X

The following procedures illustrate the syntheses of compounds in theclass defined by the structure of Compound I.

Synthesis of 6-Methoxy-3-Chlorobenzo b!thiophene-2-Carbonyl Chloride

A 100 milliliter (ml) 3-necked, round-bottomed flask, reflux condenser,thermometer adapter, gas inlet and Teflon stirrer were oven dried for 12hours at 160° C. The apparatus was rapidly assembled, fitted with athermometer, flushed with dry nitrogen gas, sealed with rubber sepia andallowed to cool to room temperature. The reaction flask was then chargedwith 4-methoxycinnamic acid (Aldrich, 10 g, 60 mmole), anhydrouspyridine (Aldrich, 1 ml, 12.4 mmole), anhydrous dimethylformamide(Aldrich 2.0 ml, 25.8 mmole) and anhydrous chlorobenzene (Aldrich, 10ml) at room temperature. At this point the apparatus was disconnectedfrom the dry nitrogen source.

Thionyl chloride (Aldrich, 205.6 mmole, 15 ml) was added to the reactionmixture in a dropwise fashion over a period of 10 minutes using asyringe. Evolution of hydrogen chloride was observed together with therapid dissolution of the white solid in the flask to give a yellow-brownsolution. Upon completion of the addition, the reaction mixture wasstirred rapidly at room temperature for 30 minutes and then heated bymeans of a heating mantle to 140°-145° C. for 5 hours. Upon cooling thereaction mixture to room temperature, the dark brown solutionsolidified.

The contents of the flask were broken into small pieces, slurried andtransferred to a 500 ml Erlenmeyer flask using hot, dry hexane (500 ml,dried over magnesium sulfate, 25 g/1000 ml). The hot hexane slurry washeated to boiling for 30 minutes during which time most of the residuegradually dissolved. The resulting orange-yellow hot hexane solution wasdecanted from the remaining residue and concentrated to 350 ml. Thesolution was allowed to cool to room temperature, and was stored at 4°C. overnight. During this period a yellow precipitate formed. Theprecipitate was filtered off and dried in vacuo to give 4.00 g of thedesired material. Further concentration of the filtrate to 200 ml andrepeating the above mentioned crystallization steps yielded anadditional 1.00 g. ¹ H NMR (in dimethylsulfoxide-d₆ (DMSO-D₆) at 400megahertz (MHz)) indicated that both samples were contaminated with4-methoxy cinnamoyl chloride. Pure material could be produced byrecrystallization from dry hexane.

Synthesis of 6-Methyl-3-Chlorobenzo b!thiophene-2-Carbonyl Chloride

A 250 ml, 3-necked, round bottomed flask, reflux condenser, thermometeradapter, gas inlet and Teflon stirrer were oven dried for 12 hours at160° C. The apparatus was rapidly assembled, fitted with a thermometer,flushed with dry nitrogen gas, sealed with rubber septa and allowed tocool to room temperature. The reaction flask was charged with4-methylcinnamic acid (Aldrich, 20 g, 123.31 mmole), anhydrous pyridine(Aldrich, 2.2 ml, 27.28 mmole), anhydrous dimethylformamide (Aldrich,4.4 ml, 56.82 mmole) and anhydrous chlorobenzene (Aldrich, 20 ml) atroom temperature. At this point the apparatus was disconnected from thedry nitrogen source.

Thionyl chloride (Aldrich, 451.32 mmole, 39.1 ml) was added to thereaction mixture in a dropwise fashion by means of a syringe, over aperiod of 20 minutes. Evolution of hydrogen chloride was observed,together with the rapid dissolution of the white solid in the flask togive a yellow-brown solution. Upon completion of addition, the reactionmixture was rapidly stirred at room temperature for 30 minutes and thenheated by means of a heating mantle to reflux for 16 hours. Upon coolingthe reaction mixture to room temperature, the dark brown solutionsolidified.

The contents of the flask were broken into small pieces, slurried andtransferred to a 1000 ml Erlenmeyer flask using hot, dry hexane (1000ml, dried over magnesium sulfate, 25 g/1000 ml). The hot hexane slurrywas heated to boiling for 30 minutes during which time, most of theresidue gradually dissolved. The resulting orange-yellow hot hexanesolution was decanted off from the remaining residue and concentrated to750 ml. The solution was allowed to cool to room temperature, and thenstored at 4° C. overnight. This resulted in a yellow precipitate, whichwas filtered off and dried in vacuo, to give 19.46 g of the desiredmaterial. ¹ H NMR (DMSO-D₆, 400 MHz) indicated that the recoveredmaterial was predominantly the desired material (a purity of greaterthan 95% was determined by NMR).

Preparation of 6-Methoxy-7,3-Dichlorobenzo b!thiophene-2-CarbonylChloride

A 100 ml, 3-necked, round bottomed flask, reflux condenser, thermometeradapter, gas inlet and Teflon stirrer were oven dried for 12 hours at160° C. The apparatus was rapidly assembled, fitted with a thermometer,flushed with dry nitrogen gas, sealed with rubber septa and cooled toroom temperature. The reaction flask was charged with 4-methoxycinnamicacid (Aldrich, 10 g, 60 mmole), anhydrous pyridine (Aldrich, 1 ml, 12. 4mmole), anhydrous dimethylformamide (Aldrich, 2.0 ml, 25.8 mmole) andanhydrous chlorobenzene (Aldrich, 10 ml) at room temperature. At thispoint the apparatus was disconnected from the dry nitrogen source.

Thionyl chloride (Aldrich, 205.6 mmole, 15 ml) was added to the reactionmixture in a dropwise fashion by means of a syringe, over a period of 10minutes. Evolution of hydrogen chloride was observed, together with therapid dissolution of the white solid in the flask to give a yellow-brownsolution. Upon completion of addition, the reaction mixture was rapidlystirred at room temperature for 30 minutes and then the mixture washeated by means of a heating mantle to a temperature of 140°-145° C. for6 days. Upon cooling the reaction mixture to room temperature, the darkbrown solution solidified.

The contents of the flask were broken into small pieces, slurried andtransferred to a 500 ml Erlenmeyer flask using hot, dry hexane (500 ml,dried over magnesium sulfate, 25 g/1000 ml). The hot hexane slurry washeated to boiling for 30 minutes during which time, most of the residuegradually dissolved. The resulting orange-yellow hot hexane solution wasdecanted from the remaining residue and the solution was concentrated to350 ml. The solution was cooled to room temperature, and then stored at4° C. overnight. A yellow precipitate formed during the cooling, whichwas filtered off, and dried in vacuo, to give 2.25 g of the desiredmaterial. Further concentration of the filtrate to 200 ml and repeatingthe above described crystallization steps yielded an additional 1.20 g.

Preparation of 6-Methoxy-7-Chlorobenzo b!thiophene-2-Carbonyl Chloride

A 100 ml, 3-necked, round bottomed flask, reflux condenser, thermometeradapter, gas inlet and Teflon stirrer were oven dried for 12 hours at160° C. The apparatus was rapidly assembled, fitted with a thermometer,flushed with dry nitrogen gas, sealed with robber septa and cooled toroom temperature. The reaction flask was charged with 4-methoxycinnamicacid (Aldrich, 10 g, 60 mmole), anhydrous pyridine (Aldrich, 1 ml, 12.4mmole), anhydrous dimethylformamide (Aldrich, 2.0 ml, 25.8 mmole) andanhydrous chlorobenzene (Aldrich, 100 ml) at room temperature. At thispoint the apparatus was disconnected from the dry nitrogen source.

Thionyl chloride (Aldrich, 480 mmole, 35 ml) was added to the reactionmixture in a dropwise fashion by means of a syringe, over a period of 10minutes. Evolution of hydrogen chloride was observed, together with therapid dissolution of the white solid in the flask to give a yellow-brownsolution. Upon completion of addition, the reaction mixture was rapidlystirred at room temperature for 30 minutes and then heated by means of aheating mantle to a temperature of 140°-145° C. for 6 days. Uponallowing the reaction mixture to cool to room temperature, the darkbrown solution solidified.

The excess thionyl chloride and chlorobenzene was removed bydistillation at reduced pressure. Upon completion of the distillation,the contents of the flask were broken-up into small pieces, slurried andtransferred to a 500 ml Erlenmeyer flask using hot, dry hexane (500 ml,dried over magnesium sulfate, 25 g/1000 ml). The hot hexane slurry washeated to boiling for 30 minutes during which time, most of the residuegradually dissolved. The resulting orange-yellow hot hexane solution wasdecanted from the remaining residue and concentrated to 350 ml. Thesolution was cooled to room temperature, and then stored at 4° C.overnight. A yellow precipitate formed which was filtered off, and driedin vacuo, to give 1.25 g of the 7-chloro b!benzothiophene.

General Preparation of Methyl, Ethyl and Isopropyl Esters from Aryl orHeteroaryl Acyl Chlorides

The corresponding carboxylic acid (10 mmoles) was transferred to a 100ml round bottomed flask. Anhydrous methanol (Aldrich, 50 ml) was addedand the acid dissolved by stirring. To this stirred solution was addedchlorotrimethylsilane (Si(CH₃)₃ Cl, 2.5 equivalents, 25 mmole). Thereaction mixture was stirred at room temperature and monitored by TLC(Silica, 1:1 hexane:ethyl acetate, and 100% ethyl acetate, iodine stainand UV visualization). Upon completion of the reaction as determined byTLC analysis (16-48 hours at room temperature), the resulting solutionwas concentrated in vacuo to give crude material, which was shown by ¹ HNMR (DMSO-D₆, 400 MHz) to be the desired material in a high level ofpurity. The crude methyl ester can be used directly or recrystallizedfrom a suitable solvent (e.g., methanol, ethanol or acetonitrile).

The preparation of the corresponding ethyl and isopropyl esters wasaccomplished using the same technique, but ethanol or isopropyl alcoholwas substituted for methanol respectively.

Preparation of Methyl 6-Methoxy-3-Chlorobenzo b!thiophene-2-Carboxylate

Crude 6-methoxy-3-chlorobenzo b!thiophene-2-carbonyl chloride (2 g, 7.67mmoles) was transferred to a 100 ml round bottomed flask. Anhydrousmethanol (Aldrich, 50 ml) was added and the acid chloride dissolved bystirring. To this stirred solution was added chlorotrimethylsilane (2.5equivalents, 19.16 mmole, 2.08 g, 2.43 ml). The reaction mixture wasstirred overnight at room temperature. TLC analysis (Silica, 1:1hexane:ethyl acetate, UV visualization) indicated complete conversion ofthe acid chloride to the methyl ester. The resulting slurry wasconcentrated in vacuo to give a yellow solid, which was shown by ¹ H NMR(DMSO-D₆, 400 MHz) to be the desired material. The crude methyl esterwas dissolved in hot ethanol (200 ml), the volume was reduced to 150 ml,and allowed to cool to room temperature. The resulting orange ethanolicsolution was cooled to -10° C. for 15 minutes. The resulting precipitatewas filtered off and dried in vacuo to give 1.25 g of the ester as anoff-white solid.

Preparation of Methyl 6-Methyl-3-Chlorobenzo b!thiophene-2-Carboxylate

Crude 6-methyl-3-chlorobenzo b!thiophene-2-carbonyl chloride (5 g, 20.46mmoles) was transferred to a 250 ml round bottomed flask. Anhydrousmethanol (Aldrich, 80 ml) was added and the acid chloride dissolved bystirring. To this stirred solution was added chlorotrimethylsilane (3.0equivalents, 61.38 mmole, 6.68 g, 7.80 ml). The reaction mixture wasstirred at room temperature and the progress of the reaction monitoredby TLC (Silica, 1:1 hexane:ethyl acetate, and 100% ethyl acetate, iodineand UV visualization). The reaction was shown to be complete after 24hours. The resulting slurry was concentrated in vacuo to give 4.80 g ofa yellow solid, which was shown by ¹ H NMR (DMSO-D₆, 400 MHz) to be thedesired material. The crude methyl ester was shown to be pure and useddirectly.

General Method for the Preparation of Aryl and Heteroaryl Hydrazidesfrom Aryl and Heteroaryl Esters

A 100 ml bottomed flask, reflux condenser, gas inlet and Teflon stirrerwere oven dried for 12 hours at 160° C. The apparatus was rapidlyassembled, flushed with dry nitrogen gas, and allowed to cool to roomtemperature. The reaction flask was charged with methyl ester (5.0mmole), anhydrous methanol (Aldrich, 50 ml), and anhydrous hydrazine(Aldrich, 2.5 equivalents, 12.5 mmole). The stirred reaction mixture wasbrought to reflux and the progress of the reaction monitored by TLC(Silica, 1:1 hexane:ethyl acetate, and 100% ethyl acetate, iodine and UVvisualization). Upon completion of the reaction, the reaction mixturewas cooled to room temperature, and further cooled to 5° C. for 1 hour.The resulting solid was filtered off, washed with cold methanol (2×10ml), and dried in vacuo. The resulting hydrazide was sufficiently pureto be used directly.

General Method for the Preparation of Aryl and Heteroaryl Hydrazidesfrom Aryl and Heteroaryl Acyl or Sulfonyl Chlorides

A 250 ml, 3-necked round bottomed flask, Teflon stirrer, 50 ml pressureequalized addition funnel, and gas inlet were oven dried for 12 hours at160° C. The apparatus was rapidly assembled, flushed with dry nitrogengas, and cooled to room temperature. The reaction flask was charged withanhydrous THF (Aldrich, 100 ml) and anhydrous hydrazine (Aldrich, 131.0mmole, 4.21 g, 4.12 ml). The reaction mixture was cooled to 0° C.(ice/water), and a THF solution (20 ml) of 2-naphthoyl chloride (10 g,52.54 moles) was added over 30 minutes in a dropwise fashion to thestirred reaction mixture. Upon completion of addition, the reactionmixture was warmed to room temperature and stirred overnight. Thereaction mixture was poured onto ice/water (1000 ml), and vigorouslystirred for 1 hour. The resulting white slurry was filtered, washed withcold water (4×50 ml), and dried in vacuo to give 9.80 g of a whitepowder. ¹ H NMR (DMSO-D₆, 400 MHz) showed the product to be pure.

Preparation of 6-Methoxy-3-Chlorobenzo b!thiophene-2-Hydrazide

A 100 ml, round bottomed flask, reflux condenser, gas inlet and Teflonstirrer were oven dried for 12 hours at 160° C. The apparatus wasrapidly assembled, flushed with dry nitrogen gas, and cooled to roomtemperature. The reaction flask was charged with methyl6-methoxy-3-chlorobenzo b!thiophene-2-carboxylate (1.50 g, 5.85 mmole),anhydrous methanol (Aldrich, 50 ml), and anhydrous hydrazine (Aldrich,2.5 equivalents, 0.47 g, 0.45 ml, 14.61 mmole). The stirred reactionmixture was brought to reflux and the progress of the reaction monitoredby TLC (Silica, 1:1 hexane:ethyl acetate, and 100% ethyl acetate, iodineand UV visualization). After 72 hours, the completed reaction was cooledto room temperature, and further cooled to 5° C. for 1 hour. Theresulting solid was filtered, washed with cold methanol (2×10 ml), anddried in vacuo. This resulted in 0.856 g of the desired material, as awhite amorphous powder which was used directly.

Preparation of 3-Chlorobenzo b!thiophene-2-Carboxy-2'-(2,5-Dichlorophenylamino)thia!hydrazine, Compound XVIII

A 50 ml round bottomed flask, Teflon stirrer and gas inlet were ovendried for 12 hours at 160° C. The apparatus was flushed with drynitrogen and cooled. The reaction flask was charged with 3-chlorobenzob!thiophene-2-carboxyhydrazide (2 g, 8.85 mmole), which was dissolvedwith anhydrous tetrahydrofuran (THF, 25 ml). To this homogeneousreaction mixture was added 2,5-dichlorophenyl isothiocyanate (1.88 g,9.2 mmole) in anhydrous THF (4 ml). The reaction mixture was stirred atroom temperature for 24 hours. The resulting slurry was diluted withanhydrous THF (10 ml), and stirred for a further 10 minutes. The solidwas filtered off, washed with 1:1 hexane-diethyl ether (15 ml), anddried in vacuo to give 1.21 g of pure product.

Preparation of 3-Chlorobenzo b!thiophene-2-Carboxy-2'-(3,4-Dichlorophenylamino)oxa!hydrazine

A 50 ml round bottomed flask, Teflon stirrer and gas inlet were ovendried for 12 hours at 160° C. The apparatus was flushed with drynitrogen and cooled. The reaction flask was charged with 3-chlorobenzob!thiophene-2-carboxyhydrazide (2 g, 8.85 mmole), which was dissolvedwith anhydrous THF (25 ml). 3,4-Dichlorophenyl isocyanate (1.73 g, 9.2mmole) in anhydrous THF (4 ml) was added in one lot to the stirredhomogeneous reaction mixture. The reaction mixture was stirred at roomtemperature for 24 hours. The resulting slurry was diluted withanhydrous THF (10 ml), and stirred for a further 10 minutes. The solidwas filtered, washed with 1:1 hexane-diethyl ether (15 ml), and dried invacuo to give 1.21 g of pure product.

2. Synthesis of Compounds XI-XLV

The following General Procedures illustrate the synthesis of thecompounds in the class defined by the structure of Compound XI.

General Procedure A: Synthesis of5-Dimethoxymethyl-3--Cyano-2-Thioetherpyridines and Pyrido b!thiophenes

The following demonstrates the synthesis of Compound 8 of Scheme 2. Intoa round-bottom flask equipped with a magnetic stir bar and under apositive pressure of nitrogen were placed3-cyano-2-mercaptopyridine-6-carboxaldehyde dimethyl acetal (1.0513 g,5.0 mmol), DMF (5 ml), K₂ CO₃ (0.7602 g, 5.5 mmol), and thecorresponding alkylating agent (5.2 mmol). The heterogeneous mixture wasstirred at room temperature for 3 hr. The reaction mixture was pouredinto ice/H₂ O (50 ml) and extracted twice with ethyl acetate (EtOAc.)The combined organic layers were washed with saturated sodium chloride(NaCl, brine), dried over magnesium sulfate (MgSO₄), filtered, and thesolvent removed by rotary evaporation to yield the coupled product.Isolated yields range from 87-96% The product was analyzed by ¹ H NMRand TLC.

General Procedure B: Acetal Deprotection of5-Dimethoxymethyl-3--Cyano-2-Thioetherpyridines and Pyrido b!thiophenes

The following demonstrates the conversion of Compound 8 of Scheme 2 intoCompound 9 of the same Scheme. Into a round-bottom, three-necked flaskequipped with a magnetic stir bar, thermometer, reflux condenser, andunder a positive pressure of nitrogen, were placed the correspondingdimethyl acetal (5.0 mmol), benzene (60 ml), and p-toluenesulfonic acid(1.9975 g, 10.5 mmol). The mixture was refluxed at 73°-78° C. for 48hours (h). The reaction was quenched by pouring the reaction mixtureinto saturated aqueous (sat. aq.) sodium bicarbonate (NaHCO₃) and thenextracted twice with methylene chloride. The combined organic layerswere washed with sat. NaCl (brine), dried over MgSO₄, filtered, and thesolvent removed by rotary evaporation to yield the desired aldehyde. Theproduct was analyzed by ¹ H NMR and TLC.

General Procedure C: Synthesis of 5-Iminio-3-Cyano-2-Thioetherpyridinesand Pyrido b!thiophenes

This procedure demonstrates the conversion of Compound 8 or Compound 9of Scheme 2 into Compound 10 of that Scheme. Into a round bottom,three-necked flask equipped with a magnetic stir bar, thermometer,reflux condenser, and under a positive pressure of nitrogen, were placedthe corresponding dimethyl acetal or aldehyde (1.0 mmol), 3 Å molecularsieves (5.0 g), benzene (12 ml), and p-toluenesulfonic acid (0.3995 g,2.1 mmol), and the amine (1.0 mmol). The mixture was refluxed at 73°-78°C. for 24-48 h. Upon cooling, the molecular sieves where filtered offand washed with methylene chloride. The filtrate was poured into sat.aq. NaHCO₃ and extracted twice with methylene chloride. The combinedorganic layers were washed with sat. NaCl (brine), dried over MgSO4 (orsodium sulfate (Na₂ SO₄)), filtered, and the solvent removed by rotaryevaporation to yield the crude product. The crude product wasrecrystallized from acetonitrile. The final product was analyzed by ¹ HNMR and TLC.

General Procedure D: Deprotection of Dimethoxyacetal Pyridine Thioethers

This procedure also illustrates the conversion of compound 8 of Scheme 2into compound 9 of that Scheme. Into a round bottom, three-necked flaskequipped with a magnetic stir bar, thermometer and under a positivenitrogen pressure, were introduced the corresponding acetal (7.0 mmol)and chloroform (CHCl₃, 63 mL). To this mixture a second mixture ofaqueous trifluoroacetic acid (TFA/H₂ O, 31 mL/31 mL) at 0° C. Themixture was stirred vigorously at 35° C. until completion, as determinedby TLC analysis (3-78 h.) The reaction was quenched with sat. aq. NaHCO₃and the solution extracted twice with EtOAc. The organic layers fromeach extraction were combined and washed with sat. aq. NaCl (brine),then dried over sodium sulfate (Na₂ SO₄) and filtered. The solvent wasremoved by rotary evaporation, and the product analyzed by ¹ H NMR andTLC.

General Procedure E: Synthesis of Hydrazide and SemicarbazideDerivatives

This procedure illustrates the formation of compound 11 of Scheme 2.Into a reaction vessel equipped with a magnetic stir bar, were placedthe corresponding dimethyl acetal (0.25 mmol), semicarbazide orhydrazide (0.25 mmol), methanol (2.5 mL), and 2 drops of concentratedhydrochloric acid (HCl.) The vessel was sealed with a rubber septa andprovided with a pressure release needle. The vessel was placed in asonicator and heated to 50° C. for two days. Upon cooling, the solidswere isolated by filtration and washed with methanol. The product wasdried overnight in a high vacuum oven.

The application of the above-described General Procedures to thesyntheses of certain compounds of the invention is illustrated in Table4 below. As noted above, the details of applying the General Proceduresto the synthesis of the individual compounds of the invention can beperformed using standard methods and materials.

                  TABLE 4                                                         ______________________________________                                        Compound  Methods     Compound  Methods                                       ______________________________________                                        XII       A, C        XXX       A, C                                          XIII      A, C        XXXI      A, C                                          XVI       A, C        XXXII     A, D, C                                       XVII      A, C        XXXIII    A, C                                          XVIII     A, C        XXXIV     A, D, C                                       XIX       A, C        XXXV      A, D, C                                       XX        A, C        XXXVI     A, C                                          XXI       A, C        XXXVII    A, C                                          XXII      A, C        XXXVIII   A, C                                          XXIII     A, C        XXXIX     A, C                                          XXIV      A, C        XLI       A, B                                          XXV       A, C        XLII      A, B                                          XXVI      A, C        XLIV      A, C                                          XXVII     A, C        XLV       A, C                                          XXVIII    A, C        XLVII     A, E                                          XXIX      A, C        XLVIII    A, E                                          ______________________________________                                    

B. Anti-tumor Activity

1. Ex vivo Studies

a. Reduction of Telomere Length in Tumor Cells

Colonies of the ovarian tumor cell lines OVCAR-5 and SK-OV-3 and normalhuman BJ cells (used as a control) were prepared using standard methodsand materials. In one test, the colonies were prepared by seeding15-centimeter dishes with about 10⁶ cells in each dish. The dishes wereincubated to allow the cell colonies to grow to about 80% confluence, atwhich time each of the colonies was divided into two groups. One groupwas exposed to a subacute dose of Compound XVIII (10 μM) 4-8 hours afterplating following the split; the other group was exposed to a DMSOcontrol.

Each group was then allowed to continue to divide, and the groups weresplit evenly again (near confluence). The same number of cells wereseeded for continued growth. The compound or control was added everyfourth day to the samples at the same concentration delivered initially(10 μM). Remaining cells were analyzed for telomere length. As theuntested cell cultures neared confluence, the samples were split againas just described. This sequence of cell doubling and splitting wascontinued for 20-25 doublings. Thus, a determination of telomere lengthas a function of cell doublings was obtained.

Telomere length was determined by digesting the DNA of the cells usingrestriction enzymes specific for sequences other than the repetitive T₂AG₃ sequence of human telomeres. The digested DNA was separated by sizeusing standard techniques of gel electrophoresis to determine thelengths of the telomeric repeats, which appeared, after probing, on thegel as a smear of high-molecular weight DNA (approximately 2 Kb-15 Kb).

The results of the telomere length analysis indicated that the testcompound of the invention had no affect on the rate of decrease intelomere length for BJ (control) cells as a function of progressive celldoublings. With respect to the tumor cell lines, however, measurabledecreases in telomere length were determined in as few as eightdoublings for tumor cells exposed to the test compound of the invention.Tumor cells exposed to the control maintained steady telomere lengths asexpected. Thus, Compound XVIII was demonstrated to cause resumption ofthe normal loss of telomere length as a function of cell division intumor cells.

In another experiment, HEK-293 cells were incubated with Compound XVIIIand a DMSO control at concentrations of 3.2 μM and 10 μM using theprotocol just described. Cells were observed to enter crisis (i.e., thecessation of cell function) within 12 weeks following administration ofCompound XVIII at 10 μM and after 16 weeks in the case where CompoundXVIII was administered at a concentration of 3.2 μM. In addition, TRFanalyses of the cells using standard methodology showed that the testcompound caused reductions in telomere length by 350 bp and 330 bp,respectively. No cytotoxic effects were observed.

b. Specificity

Compound XVIII was screened for activity (IC₅₀) against telomerase andseveral enzymes having nucleic acid binding or modifying activitiesrelated to telomerase using standard techniques. The enzymes screenedincluded Telomerase, DNA Polymerase I, HeLa RNA Polymerase II, T3 RNAPolymerase, MMLV Reverse Transcriptase, Topoisomerase I, TerminalTransferase and Single-Stranded DNA Binding Protein (SSB). CompoundXVIII was also found to have high specificity for telomerase as comparedto Glucose-6-Phosphate Dehydrogenase. The test compound was found tohave an IC₅₀ of 30 μM with respect to telomerase and an IC₅₀ of 100 μMwith respect to SSB. No significant inhibition was observed with respectto any of the remaining enzymes. Thus, Compound XVIII demonstrated highspecificity for telomerase.

c. Cytotoxicity

The MTT assay for cytotoxicity was performed using the ovarian tumorcells lines OVCAR-5 and SK-OV-3. Cells from the normal human cell lineBJ were used as a control. The cell lines used in the assay were exposedto Compound XVIII of the invention for 72 hours at concentrationsranging from 3 μM to 1,000 μM. During this period, the optical density(OD) of the samples was determined for light at 540 nanometers (nm). Nosignificant cytotoxic effects were observed at concentrations less thanabout 15 μM.

2. In vivo Studies

A human tumor xenograft model in which OVCAR-5 tumor cells are graftedinto nude mice can be constructed using standard techniques andmaterials. The mice are divided into two groups. One group is treatedintraperitoneally with Compound XVIII. The other group is treated with acontrol comprising a mixture of either DMSO or ethanol and emulphor(oil) and phosphate buffer solution (PBS). The average tumor mass formice in each group is determined periodically following the xenograftusing standard methods and materials.

In the group treated with a compound of the invention (e.g., CompoundXVIII), the average tumor mass is expected to increase following theinitial treatment for a period of time, after which time the tumor massis expected to stabilize and then begin to decline. Tumor masses in thecontrol group are expected to increase throughout the study. Thus, thecompounds of the invention are expected to lessen dramatically the rateof tumor growth and ultimately induce reduction in tumor size andelimination of the tumor.

Thus, the present invention provides novel compounds, compositions andmethods for inhibiting telomerase activity and treating disease statesin which telomerase activity has deleterious effects, especially cancer.The compounds of the invention provide a highly selective and effectivetreatment for malignant cells that require telomerase activity to remainimmortal; yet, without affecting non-malignant cells.

Although certain embodiments and examples have been used to describe thepresent invention, it will be apparent to those of skill in the art thatchanges may be made to those described embodiments and examples withoutdeparting from the scope or spirit of the invention or the followingclaims.

What is claimed:
 1. A telomerase inhibiting compound having thestructure: ##STR28## wherein R₂ is hydrogen or halogen; R₃ -R₆ areselected independently from the group consisting of hydrogen, halogen,hydroxyl, --NR₈ R₉, nitro, cyano, alkoxyl, lower alkyl, aryl andaryloxyl, where R₈ and R₉ are selected independently from the groupconsisting of hydrogen, alkyl, aryl, aralkyl, heteroaryl, andheteroaralkyl; R₁₂ is selected from the group consisting of hydrogen,alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl; and X₁ and X₂ areselected independently from the group consisting of oxygen andsulfur;provided that when X₁ is oxygen, X₂ is sulfur, and R₂ is chloro:R₁₂ is not methyl, phenyl, 4-methoxyphenyl or 4-chlorophenyl when R₃,R₄, and R₆, are hydrogen and R₅ is methoxy; and R₁₂ is not methyl,phenyl, 4-methoxyphenyl, 4-chlorophenyl, 4-methylphenyl,2,5-dichlorophenyl, phenylmethyl, or unsubstituted allyl when R₃ -R₆ arehydrogen.
 2. The compound of claim 1 wherein R₁₂ is aryl.
 3. Thecompound of claim 2 having the structure ##STR29## wherein R₁₃ -R₁₇ areselected independently from the group consisting of hydrogen, halogen,hydroxyl, alkylthio, arylthio, nitro, cyano, amino, alkylamino,arylamino, dialkylamino, diarylamino, arylalkylamino, alkoxyl, alkyl,aryl, aralkoxyl, aralkylthio, arylcarbonyloxy, and aryloxyl.
 4. Thecompound of claim 3 wherein R₃ -R₆ are selected independently from thegroup consisting of hydrogen, chloro, bromo, fluoro, trifluoromethyl,dimethylamino, nitro, methyl, methoxy, phenyl, and aryloxyl.
 5. Thecompound of claim 4, wherein X₁ is oxygen, X₂ is sulfur, and R₂ ischloro.
 6. The compound of 5, wherein R₁₃ -R₁₇ are selectedindependently from the group consisting of hydrogen, chloro, bromo,fluoro, methyl, trifluoromethyl, dimethylamino, 4-methylphenylthio,4-nitrophenylthio and methylthio.
 7. The compound of claim 6, wherein R₃-R₆ are selected independently from the group consisting of hydrogen,chloro, bromo, fluoro, and trifluoromethyl.
 8. The compound of claim 7,wherein R₁₃ -R₁₇ are selected independently from the group consisting ofhydrogen, chloro, bromo, fluoro, and trifluoromethyl.
 9. The compound ofclaim 8, wherein R₃ -R₆ are selected independently from the groupconsisting of hydrogen and chloro.
 10. The compound of claim 9, whereinR₁₃ -R₁₇ are selected independently from the group consisting ofhydrogen and chloro.
 11. The compound of claim 4, wherein X₁ is oxygen,X₂ is sulfur, and R₂ is hydrogen.
 12. The compound of 11, wherein R₁₃-R₁₇ are selected independently from the group consisting of hydrogen,chloro, bromo, fluoro, methyl, trifluoromethyl, dimethylamino,4-methylphenylthio, 4-nitrophenylthio and methylthio.
 13. The compoundof claim 12, wherein R₃ -R₆ are selected independently from the groupconsisting of hydrogen, chloro, bromo, fluoro, and trifluoromethyl. 14.The compound of claim 13, wherein R₁₃ -R₁₇ are selected independentlyfrom the group consisting of hydrogen, chloro, bromo, fluoro, andtrifluoromethyl.
 15. The compound of claim 14, wherein R₃ -R₆ areselected independently from the group consisting of hydrogen and chloro.16. The compound of claim 15, wherein R₁₃ -R₁₇ are selectedindependently from the group consisting of hydrogen and chloro.
 17. Thecompound of claim 10 which is: ##STR30##
 18. The compound of claim 10which is: ##STR31##
 19. The compound of claim 10 which is: ##STR32## 20.The compound of claim 10 which is: ##STR33##
 21. A composition fortreating cancer, comprising a therapeutically effective amount of acompound having the structure shown below in a pharmaceuticallyacceptable carrier: ##STR34## wherein R₂ is hydrogen or halogen; R₃ -R₆are selected independently from the group consisting of hydrogen,halogen, hydroxyl, --NR₈ R₉, nitro, cyano, alkoxyl, lower alkyl, aryland aryloxyl, where R₈ and R₉ are selected independently from the groupconsisting of hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroalkyl,alkylsulfonyl, arylsulfonyl, arylcarbonyl, and alkylcarbonyl; R₁₂ isselected from the group consisting of hydrogen, alkyl, aryl, aralkyl,heteroaryl, and heteroaralkyl; and X₁ and X₂ are selected independentlyfrom the group consisting of oxygen and sulfur.
 22. A method of treatingcancer in a mammal, comprising administering to such mammal atherapeutically effective amount of a compound having the structureshown in claim 21 to inhibit telomerase activity in cancer cells is saidmammal such that the telomeres of said cancer cells are reduced inlength over successive cell divisions.